Identification of a novel bitter taste receptor, T2R76

ABSTRACT

Isolated nucleic acids encoding T2R76 polypeptides, recombinantly expressed T2R76 polypeptides, heterologous expression systems for recombinant expression of T2R76 polypeptides, assay methods employing the same, and methods for altering taste perception via administration of a T2R76 modulator. These T22R76 polypeptides can be expressed alone or co-expressed with another T2R polypeptide, preferably a different human T2R polypeptide.

RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Ser. No. 60/398,727filed on Jul. 29, 2002 which Application is incorporated by reference inits entirety.

FIELD OF THE INVENTION

[0002] The present invention generally relates to T2R76 polypeptides andtaste perception mediated by the same. More particularly, the presentinvention provides isolated nucleic acids encoding T2R76 polypeptides,isolated and functional T2R76 polypeptides, a heterologous expressionsystem for recombinant expression of T2R76 polypeptides, methods foridentifying modulators of taste perception, especially compounds whichare bitter tasting or which block bitter taste, and uses thereof.

DESCRIPTION OF RELATED ART

[0003] One of the basic taste modalities that humans can recognize isbitter. Bitter compounds are thought to produce bitter taste byinteracting with cell surface receptors. Activation of the receptorsinitiates intracellular signaling cascades that culminate inneurotransmitter release. Afferent nerve fibers from cranial nerveganglia then relay the signals to cortical taste centers, where theinformation is processed as taste perception. These receptors belong tothe family of seven transmembrane domain receptors that interact withintracellular G proteins, also called G protein-coupled receptors(GPCRs). See Lindemann (2001) Nature 413(6852): 219-25.

[0004] A novel family of GPCRs, termed T2Rs, has been identified inhumans and rodents (Adler et al., 2000; Chandrashekar et al., 2000;Matsunami, 2000; PCT International Publication Nos. WO 01/18050 and WO01/77676). Several lines of evidence suggested that the T2Rs can mediateperception of bitter compounds. First, the T2R genes are specificallyexpressed in subset of taste receptor cells of the tongue and palateepithelia. Second, T2Rs are genetically linked to loci associated withbitter perception in mice and humans (Conneally et al., 1976; Capelesset al., 1992; Reed et al., 1999; Adler et al., 2000). Third, in vitrostudies have shown that T2Rs can activate gustducin, a G proteinspecifically expressed in taste cells and linked to bitter stimulitransduction (Wong et al., 1996), and that gustducin activation by T2Rsoccurs selectively in response to the application of bitter compounds(Chandrashekar et al., 2000). Based on these data, the mT2R and hT2Rreceptor families are proposed to mediate bitter taste response in miceand human, respectively.

[0005] Bitter tastes are often undesirable in food, beverages, oralwashes, dentifrices, cosmetics, and pharmaceuticals. A bitter taste canbe masked by the addition of sweet compounds, such as sugar; however,the addition of a sweetener may undesirably alter a food flavor andincrease calorie intake. In the case of pharmaceuticals, elaborate andcostly formulation methods (e.g., coatings and capsules) have beendeveloped to reduce bitter taste upon oral intake. Methods for directlyblocking bitter taste via inhibition of taste receptors have not beendescribed.

[0006] Thus, there exists a long-felt need in the art to identify andfunctionally characterize bitter taste receptors as targets for thedevelopment of inhibitors of bitter taste perception. To meet this need,the present invention provides novel T2R76 nucleic acids andpolypeptides. The present invention also provides methods foridentifying and using modulators of T2R76 to alter taste perception.

SUMMARY OF INVENTION

[0007] The present invention provides isolated T2R76 nucleic acids andT2R76 polypeptides encoded by the same. The polypeptides and nucleicacids are useful in the detection methods and assays disclosed herein.

[0008] A T2R76 nucleic acid can comprise: (a) an isolated nucleic acidmolecule encoding a polypeptide of SEQ ID NO:2; (b) an isolated nucleicacid molecule of SEQ ID NO:1; or (c) an isolated nucleic acid moleculesubstantially similar to SEQ ID NO:1.

[0009] A TR76 nucleic acid can also comprise: (a) an isolated nucleicacid molecule encoding a polypeptide of SEQ ID NO:2; (b) an isolatednucleic acid molecule of SEQ ID NO: 1; (c) an isolated nucleic acidmolecule which hybridizes to a nucleic acid sequence of SEQ ID NO:1under wash stringency conditions represented by a wash solution havingless than about 200 mM salt concentration and a wash temperature ofgreater than about 45° C., and which encodes a T2R76 polypeptide; or (d)an isolated nucleic acid molecule differing by at least one functionallyequivalent codon from the isolated nucleic acid molecule of one of (a),(b), and (c) above in nucleic acid sequence due to the degeneracy of thegenetic code, and which encodes a T2R76 polypeptide encoded by theisolated nucleic acid of one of (a), (b), and (c) above. Preferably, anisolated T2R76 nucleic acid comprises: (a) an isolated nucleic acidmolecule encoding a polypeptide of SEQ ID NO:2; or (b) an isolatednucleic acid molecule of SEQ ID NO:1.

[0010] An isolated T2R76 polypeptide can comprise: (a) a polypeptide ofSEQ ID NO:2; (b) a polypeptide substantially identical to SEQ ID NO:2;(c) a polypeptide encoded by a nucleic acid molecule of SEQ ID NO:1; or(d) a polypeptide encoded by a nucleic acid molecule substantiallyidentical to SEQ ID NO:1.

[0011] A T2R76 polypeptide can also comprise a polypeptide encoded by anisolated nucleic acid molecule selected from the group consisting of:(a) an isolated nucleic acid molecule encoding a polypeptide of SEQ IDNO:2; (b) an isolated nucleic acid molecule of SEQ ID NO:1; (c) anisolated nucleic acid molecule that hybridizes to a nucleic acid of SEQID NO:1 under high stringency conditions, and that encodes a T2R76polypeptide; and (d) an isolated nucleic acid molecule differing by atleast one functionally equivalent codon from the isolated nucleic acidmolecule of one of (a), (b), or (c) above in nucleic acid sequence dueto the degeneracy of the genetic code, and which encodes a T2R76polypeptide encoded by the isolated nucleic acid of (a), (b), or (c)above. Preferably, a T2R76 polypeptide comprises SEQ ID NO:2.

[0012] The present invention further provides methods for detecting aT2R76 nucleic acid, the method comprising: (a) procuring a biologicalsample having nucleic acid material; (b) hybridizing an isolated T2R76nucleic acid molecule under stringent hybridization conditions to thebiological sample of (a), thereby forming a duplex structure between theisolated T2R76 nucleic acid and a nucleic acid within the biologicalsample; and (c) detecting the duplex structure of (b), whereby a T2R 76nucleic acid molecule is detected.

[0013] The present invention further provides antibodies thatspecifically recognize a T2R76 polypeptide, and methods for producingthe same. A representative embodiment of the method comprises: (a)recombinantly or synthetically producing a T2R76 polypeptide; (b)formulating the polypeptide of (a) whereby it is an effective immunogen;(c) administering to an animal the formulation of (b) to generate animmune response in the animal comprising production of antibodies,wherein antibodies are present in the blood serum of the animal; and (d)collecting the blood serum from the animal of (c) comprising antibodiesthat specifically recognize a T2R76 polypeptide. The disclosed methodcan further comprise preparing a monoclonal antibody.

[0014] Also provided are methods for detecting a level of a T2R76polypeptide. In a representative embodiment, the method comprises: (a)obtaining a biological sample having peptidic material; (b) detecting aT2R76 polypeptide in the biological sample of (a) by immunochemicalreaction with the antibody of the present invention, whereby an amountof T2R76 polypeptide in a sample is determined.

[0015] Also provided are systems for recombinant expression of a T2R76polypeptide. A recombinant expression system can comprise: (a) a T2R76polypeptide of the invention (e.g., a representative embodiment setforth as SEQ ID NO:2); and (b) a heterologous host cell expressing theT2R76 polypeptide. Additionally, the recumbinant expression system cancomprise nucleic acid sequences encoding different T2R polypeptides thanT2R76. In particular, the recumbinant espression system may include anyof the T2R nucleic acid sequences disclosed in U.S. Pat. No. 6,558,910issued on May 6, 2003 to Zuker et al, U.S. published application Ser.No. 20/020,094,551, by Adler, John Elliot published Jul. 18, 2002, andU.S. published application Ser. No. 20/030,022,278 by Zuker et al.,published on Jan. 30, 2003, all of which are incorporated by referencein their entirety. It should be noted that another name for T2Rpolypeptides is SF or GR polypeptides, as disclosed in the ZukerApplications incorporated by reference herein. The subject hT2R76 may beexpressed with one or more other T2R polypeptides to produce afunctional heteromenic taste receptor. The other T2R polypeptides may beanother human T2R or T2R of another species, e.g., rat or mouse. A hostcell can comprise any suitable cell. A preferred host cell comprises amammalian cell, more preferably a human cell. Also preferably, a hostcell comprises a G protein alpha subunit capable of coupling to a T2R76polypeptide, for example, a promiscuous G protein such as Gα15,gustducin or transducin.

[0016] Using the disclosed system for recombinant expression of a T2R76polypeptide, the present invention further provides a method foridentifying modulators of a T2R76 polypeptide. In a preferred embodimentof the invention, the method comprises: (a) providing a recombinantexpression system whereby a T2R76 polypeptide is expressed in aheterologous host cell; (b) providing a test substance to the system of(a); (c) assaying a level or quality of T2R76 function in the presenceof the test substance; (d) comparing the level or quality of T2R76function in the presence of the test substance with a control level orquality of T2R76 function; and (e) identifying a test substance as aT2R76 modulator by determining a level or quality of T2R76 function inthe presence of the test substance as significantly changed whencompared to a control level or quality of T2R76 function. The assayingcan comprise determining an amount of GTPγS binding.

[0017] In another embodiment of the invention, a method for identifyinga modulator of a T2R76 polypeptide comprises: (a) expressing a T2R76polypeptide and expressing said polypeptide or polypeptide combinationsalone or in combination with one or more other T2R polypeptides to oneor more test substances; (b) assaying binding of a test substance to theisolated T2R76 polypeptide or T2R76 containing polypeptide combination;and (c) selecting a candidate substance that demonstrates specificbinding to the T2R76 polypeptide.

[0018] Also provided are modulators, including agonists and inhibitorsof a T2R76 polypeptide, that are identified by the disclosed methods. Amodulator can comprise a protein, a peptide, an antibody, a nucleicacid, a small molecule, or combinations thereof. Preferably, a modulatorfurther comprises a modulator of bitter taste perception.

[0019] The present invention further provides methods for modulatingbitter taste perception in a subject. Preferably, the subject is amammalian subject, and more preferably a human subject. Also preferably,the bitter taste perception that is altered in a subject comprises aT2R76 function.

[0020] In one embodiment of the present invention, a method formodulating bitter taste perception in a subject comprises: (a) preparinga composition comprising a T2R76 modulator identified according to thedisclosed methods; and (b) administering an effective dose of thecomposition to a subject, whereby bitter taste perception in the subjectis altered.

[0021] For example, the present invention provides methods for reducingbitter taste perception of a bitter compound via co-administering aT2R76 inhibitor and the bitter compound to a subject. The presentinvention also provides methods for enhancing bitter taste perception ofa compound via co-administering a T2R76 agonist and the compound. Theco-administering can comprise administering a composition comprising theT2R76 inhibitor admixed with the compound whose taste is to bemodulated. In preferred embodiments of the invention, the compositioncan comprise a food, a beverage, an oral wash, a dentifrice, a cosmetic,or a pharmaceutical.

[0022] The present invention also provides methods for enhancing bittertaste perception of a compound via co-administering a T2R76 agonist andthe compound whose taste is to be modulated. The T2R76 agonist and thecompound can be admixed as a single composition.

[0023] Accordingly, it is an object of the present invention to providenovel T2R76 nucleic acids and polypeptides, methods for detecting aT2R76 nucleic acid, heterologous expression systems whereby a T2R76polypeptide is expressed, methods and assays employing a heterologousT2R76 expression system, and methods for modulating and detecting aT2R76 polypeptide. This object is achieved in whole or in part by thepresent invention.

[0024] An object of the invention having been stated above, otherobjects and advantages of the present invention will become apparent tothose skilled in the art after a study of the following description ofthe invention and non-limiting Examples.

BRIEF DESCRIPTION OF SEQUENCES IN THE SEQUENCE LISTING

[0025] [SEQ ID No: 1 and 2 are human T2R76 nucleotide and amino acidsequences, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Definitions

[0027] While the following terms are believed to be well understood byone of ordinary skill in the art, the following definitions are setforth to facilitate explanation of the invention.

[0028] The terms “a,” “an,” and “the” are used in accordance withlong-standing convention to refer to one or more.

[0029] The term “about”, as used herein when referring to a measurablevalue such as a percentage of sequence identity (e.g., when comparingnucleotide and amino acid sequences as described herein below), anucleotide or protein length, an amount of binding, etc. is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedamount, as such variations are appropriate to perform a disclosed methodor otherwise carry out the present invention.

[0030] II. T2R76 Nucleic Acids and Polypeptides

[0031] The present invention provides novel T2R76 nucleic acids andnovel T2R76 polypeptides, including functional T2R76 polypeptides. Arepresentative T2R76 nucleic acid of the present invention is set forthas SEQ ID NO:1, which encodes the T2R76 polypeptide set forth as SEQ IDNO:2.

[0032] The term “T2R76” and terms including “T2R76” (e.g., hT2R76) refergenerally to isolated T2R76 nucleic acids, isolated polypeptides encodedby T2R76 nucleic acids, and activities thereof. T2R76 nucleic acids andpolypeptides can be derived from any organism. The terms “T2R76” andterms including “T2R76” also refer to polypeptides comprising receptorsthat are activated by bitter compounds, and to nucleic acids encodingthe same. A T2R76 receptor may comprise other T2R polypeptides, and itmay be a heteromenic receptor.

[0033] The term “isolated”, as used in the context of a nucleic acid orpolypeptide, indicates that the nucleic acid or polypeptide exists apartfrom its native environment and is not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form or can exist ina non-native environment such as a transgenic host cell.

[0034] As disclosed further herein below, the present invention alsoprovides a system for functional expression of a T2R76 polypeptide. Thesystem employs a recombinant T2R76 nucleic acid, including SEQ ID NO: 1,which may be expressed in association with another T2R nucleic acid.

[0035] II.A. T2R76 Nucleic Acids

[0036] The terms “nucleic acid molecule” and “nucleic acid” each referto deoxyribonucleotides or ribonucleotides and polymers thereof insingle-stranded, double-stranded, or triplexed form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides that have similar properties as the referencenatural nucleic acid. The terms “nucleic acid molecule” or “nucleicacid” can also be used in place of “gene,” “cDNA,” “mRNA,” or “cRNA.”Nucleic acids can be synthesized, or can be derived from any biologicalsource, including any organism. Representative methods for cloning afull-length T2R76 cDNA are described in Example 1.

[0037] The term “T2R” or “SF” refers to nucleic acids encoding member ofa family of taste=cell specific G protein _______ receptor. Thesenucleic acids and the polpeptides they encode are referred to as the“T2R”, “SF” “GR”, or TAS2R family of G-protein optical taste receptors.This neuril family of GPCRs includes components of the tastetransduction p_______. For family ________, members of this family areinvolved in the detection of bitter tastes. Members of the T2R or SFfamily of taste receptors are discussed in U.S. Pat. No. 6,558,910;published U.S. patent application Ser. No. 20/030,022,278 by Zura etal., published Jan. 20, 2003; and published U.S. patent application Ser.No. 20/020,094,502, by Adler, Jon Elliot, published Jul. 18, 2002.Examples of such T2Rs include Gro1 (SF01); GR02 (SF02); GR02 (SF03);GR04 (SF04); GR05 (SF05); GR06 (SF06); GR07 (SF07); GR08 (SF08); (GR09(SF09); GR10 (SF10); GRIL (SF11); GR12 (SF12); GR13 (SF13); GR14 (SF14);GR15 (SF15); GR16 (SF16); GR17 (SF17); GR18 (SF18); GR19 (SF19); GR20(SF20); GR21 (SF21); GR22 (SF23); GR24 (SF24);

[0038] T2R51; T2R55; T2R33; T2R59; T2R61; T2R63; T2R64; T2R65; T2R75;GR25 (SF25); GR26 (SF26); GR27 (SF27); GR28 (SF28); GR29 (SF29); GR30(SF30); GR31 (SF31); GR32 (SF32); GR(SF); GR 33 (SF33); GR 34(SF24);GR35 (SF35); GR36 (SF36); GR37 (SF37); GR38 (SF38); GR39 (SF39); GR40(SF40); GR41 (SF41); GR42 (SF42); GR43 (SF43); GR44 (SF44); GR45 (SF45);GR46 (SF46); GR47 (SF47); GR48 (SF48); GR(SF); GR49 (SF49); GR50 (SF50);

[0039] These T2Rs, SFs, TAS2Rs, et al. or GRs as they are alternativelyreferred to may be of different species, including human, mouse and rat,and preferably are human. Also encompassed are T2Rs that are“substantially identical” or which possess a specific sequence identitytherewith , or which specifically hybridize to any of these sequences asdefined infra.

[0040] The terms “T2R76” and terms including “T2R76” (e.g., hT2R76) areused herein to refer to nucleic acids that encode a T2R76 polypeptide.Thus, the term “T2R76” refers to isolated nucleic acids of the presentinvention comprising:

[0041] (a) a nucleotide sequence comprising the nucleotide sequence ofSEQ ID NO:1; or

[0042] (b) a nucleotide sequence substantially identical to SEQ ID NO:1.

[0043] The term “substantially identical”, as used herein to describe adegree of similarity between nucleotide sequences, refers to two or moresequences that have at least about least 60%, preferably at least about70%, more preferably at least about 80%, more preferably about 90% toabout 99%, still more preferably about 95% to about 99%, and mostpreferably about 99% nucleotide identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists in nucleotide sequences of at least about100 residues, more preferably in nucleotide sequences of at least about150 residues, and most preferably in nucleotide sequences comprising afull length coding sequence. The term “full length” is used herein torefer to a complete open reading frame encoding a functional T2R76polypeptide, as described further herein below. Methods for determiningpercent identity between two polypeptides are defined herein below underthe heading “Nucleotide and Amino Acid Sequence Comparisons”.

[0044] In one aspect, substantially identical sequences can bepolymorphic sequences. The term “polymorphic” refers to the occurrenceof two or more genetically determined alternative sequences or allelesin a population. An allelic difference can be as small as one base pair.

[0045] In another aspect, substantially identical sequences can comprisemutagenized sequences, including sequences comprising silent mutations.A mutation can comprise one or more residue changes, a deletion ofresidues, or an insertion of additional residues.

[0046] Another indication that two nucleotide sequences aresubstantially identical is that the two molecules hybridize specificallyto or hybridize substantially to each other under stringent conditions.In the context of nucleic acid hybridization, two nucleic acid sequencesbeing compared can be designated a “probe” and a “target.” A “probe” isa reference nucleic acid molecule, and a “target” is a test nucleic acidmolecule, often found within a heterogeneous population of nucleic acidmolecules. A “target sequence” is synonymous with a “test sequence.”

[0047] A preferred nucleotide sequence employed for hybridizationstudies or assays includes probe sequences that are complementary to ormimic at least an about 14 to 40 nucleotide sequence of a nucleic acidmolecule of the present invention. Preferably, probes comprise 14 to 20nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100,200, 300, or 500 nucleotides or up to the full length of any SEQ IDNO:1. Such fragments can be readily prepared by, for example, chemicalsynthesis of the fragment, by application of nucleic acid amplificationtechnology, or by introducing selected sequences into recombinantvectors for recombinant production.

[0048] The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

[0049] The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

[0050] “Stringent hybridization conditions” and “stringent hybridizationwash conditions” in the context of nucleic acid hybridizationexperiments such as Southern and Northern blot analysis are bothsequence- and environment-dependent. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes, part I chapter 2, Elsevier, New York, N.Y.Generally, highly stringent hybridization and wash conditions areselected to be about 50° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Typically,under “stringent conditions” a probe will hybridize specifically to itstarget subsequence, but to no other sequences.

[0051] The Tm is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the Tm fora particular probe. An example of stringent hybridization conditions forSouthern or Northern Blot analysis of complementary nucleic acids havingmore than about 100 complementary residues is overnight hybridization in50% formamide with 1 mg of heparin at 42° C. An example of highlystringent wash conditions is 15 minutes in 0.1×SSC at 65° C. An exampleof stringent wash conditions is 15 minutes in 0.2×SSC buffer at 65° C.See Sambrook et al., eds (1989) Molecular Cloning: A Laborato[y Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. for adescription of SSC buffer. Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An example ofmedium stringency wash conditions for a duplex of more than about 100nucleotides, is 15 minutes in 1×SSC at 45° C. An example of lowstringency wash for a duplex of more than about 100 nucleotides, is 15minutes in 4× to 6×SSC at 40° C. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1 M Na+ ion, typically about 0.01 to 1 M Na⁺ ionconcentration (or other salts) at pH 7.0-8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2-fold (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

[0052] The following are examples of hybridization and wash conditionsthat can be used to identify nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: aprobe nucleotide sequence preferably hybridizes to a target nucleotidesequence in 7% sodium dodecyl sulphate (SDS), 0.5M NaP04, I mM EDTA at50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulphate (SDS), 0.5M NaP04, 1 mM EDTA at 50° C. followed by washing in1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequencehybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaP04, 1 mM EDTA at50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodiumdodecyl-sulphate (SDS), 0.5M NaP04, 1 mM EDTA at 50° C. followed bywashing in O.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe andtarget sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5MNaP04, 1 mM EDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at65° C.

[0053] A further indication that two nucleic acid sequences aresubstantially identical is that proteins encoded by the nucleic acidsare substantially identical, share an overall three-dimensionalstructure, or are biologically functional equivalents. These terms aredefined further under the heading “T2R76 Polypeptides” herein below.Nucleic acid molecules that do not hybridize to each other understringent conditions are still substantially identical if thecorresponding proteins are substantially identical. This can occur, forexample, when two nucleotide sequences comprise conservativelysubstituted variants as permitted by the genetic code.

[0054] The term “conservatively substituted variants” refers to nucleicacid sequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues. See Batzer et al. (1991)Nucleic Acids Res 19:5081; Ohtsuka et al. (1985) J Biol Chem260:2605-2608; and Rossolini et al. (1994) Mol Cell Probes 8:91-98.

[0055] The term “T2R” also encompasses nucleic acids comprisingsubsequences and elongated sequences of a T2R nucleic acid, preferablyT2R76 including nucleic acids complementary to a T2R nucleic acid, T2RRNA molecules, and nucleic acids complementary to T2R RNAs (cRNAs).

[0056] The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein above, or a primer. The term“primer” as used herein refers to a contiguous sequence comprising about8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20nucleotides, and more preferably 20-30 nucleotides of a selected nucleicacid molecule. The primers of the invention encompass oligonucleotidesof sufficient length and appropriate sequence so as to provideinitiation of polymerization on a nucleic acid molecule of the presentinvention.

[0057] The term “elongated sequence” refers to an addition ofnucleotides (or other analogous molecules) incorporated into the nucleicacid. For example, a polymerase (e.g., a DNA polymerase) can addsequences at the 3′ terminus of the nucleic acid molecule. In addition,the nucleotide sequence can be combined with other DNA sequences, suchas promoters, promoter regions, enhancers, polyadenylation signals,intronic sequences, additional restriction enzyme sites, multiplecloning sites, and other coding segments.

[0058] The term “complementary sequences,” as used herein, indicates twonucleotide sequences that comprise antiparallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison methods set forth below,or is defined as being capable of hybridizing to the nucleic acidsegment in question under relatively stringent conditions such as thosedescribed herein. A particular example of a complementary nucleic acidsegment is an antisense oligonucleotide.

[0059] The present invention also provides chimeric genes comprising thedisclosed T2R76 nucleic acids and recombinant T2R76 nucleic acids. Thus,also included are constructs and vectors comprising T2R76 nucleic acids,optionally expressed in combination with other T2R nucleic acids..

[0060] The term “gene” refers broadly to any segment of DNA associatedwith a biological function. A gene encompasses sequences including butnot limited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment that is a specific recognitionsequence for regulatory proteins, a non-expressed DNA segment thatcontributes to gene expression, a DNA segment designed to have desiredparameters, or combinations thereof. A gene can be obtained by a varietyof methods, including cloning from a biological sample, synthesis basedon known or predicted sequence information, and recombinant derivationof an existing sequence.

[0061] The term “chimeric gene,” as used herein, refers to a promoterregion operatively linked to a T2R sequence, e.g., a T2R cDNA, a T2Rnucleic acid encoding an antisense RNA molecule, a T2R nucleic acidencoding an RNA molecule having tertiary structure (e.g., a hairpinstructure) or a T2R nucleic acid encoding a double-stranded RNAmolecule. The term “chimeric gene” also refers to a T2R promoter regionoperatively linked to a heterologous sequence. Preparation of a chimericgene of the present invention is described in Example 2. Preferably, theT2R is T2R76.

[0062] The term “operatively linked”, as used herein, refers to afunctional combination between a promoter region and a nucleotidesequence such that the transcription of the nucleotide sequence iscontrolled and regulated by the promoter region. Techniques foroperatively linking a promoter region to a nucleotide sequence are knownin the art.

[0063] The term “recombinant” generally refers to an isolated nucleicacid that is replicable in a non-native environment. Thus, a recombinantnucleic acid can comprise a non-replicable nucleic acid in combinationwith additional nucleic acids, for example vector nucleic acids, thatenable its replication in a host cell.

[0064] The term “vector” is used herein to refer to a nucleic acidmolecule having nucleotide sequences that enable its replication in ahost cell. A vector can also include nucleotide sequences to permitligation of nucleotide sequences within the vector, wherein suchnucleotide sequences are also replicated in a host cell. Representativevectors include plasmids, cosmids, and viral vectors. A vector can alsomediate recombinant production of a T2R76 polypeptide, as describedfurther herein below.

[0065] The term “construct”, as used herein to describe a type ofconstruct comprising an expression construct, refers to a vector furthercomprising a nucleotide sequence operatively inserted with the vector,such that the nucleotide sequence is recombinantly expressed.

[0066] The terms “recombinantly expressed” or “recombinantly produced”are used interchangeably to refer generally to the process by which apolypeptide encoded by a recombinant nucleic acid is produced.

[0067] Thus, preferably recombinant T2R, nucleic acides, i.e., T2R76nucleic acids comprise heterologous nucleic acids. The term“heterologous nucleic acids” refers to a sequence that originates from asource foreign to an intended host cell or, if from the same source, ismodified from its original form. A heterologous nucleic acid in a hostcell can comprise a nucleic acid that is endogenous to the particularhost cell but has been modified, for example by mutagenesis or byisolation from native cis-regulatory sequences. A heterologous nucleicacid also includes non-naturally occurring multiple copies of a nativenucleotide sequence. A heterologous nucleic acid can also comprise anucleic acid that is incorporated into a host cell's nucleic acids at aposition wherein such nucleic acids are not ordinarily found.

[0068] Nucleic acids of the present invention can be cloned,synthesized, altered, mutagenized, or combinations thereof. Standardrecombinant DNA and molecular cloning techniques used to isolate nucleicacids are known in the art. Site-specific mutagenesis to create basepair changes, deletions, or small insertions are also known in the art.See e.g., Sambrook et al. (eds.) (1989) Molecular Cloning LaboratoryManual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Silhavy et al. (1984) Experiments with Gene Fusions. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover & Hames (1995) DNACloning: A Practical Approach, 2 nd ed. IRL Press at Oxford UniversityPress, Oxford/New York; Ausubel (ed.) (1995) Short Protocols inMolecular Biology, 3 rd ed. Wiley, New York.

[0069] III.B. T2R76 Pollypeptides

[0070] The present invention provides novel T2R76 polypeptides, arepresentative embodiment of which is set forth as SEQ ID NOs:2.Preferably, an isolated T2R76 polypeptide of the present inventioncomprises a recombinantly expressed T2R76 polypeptide. Also preferably,isolated T2R76 polypeptides comprise functional T2R76 polypeptides.These T2R76 polypeptides may be expressed in combination with one ormore other T2R polypeptides.

[0071] Thus, novel T2R76 polypeptides useful in the methods of thepresent invention comprise: (a) a polypeptide of SEQ ID NO:2; (b) apolypeptide substantially identical to SEQ ID NO:2; (c) a polypeptideencoded by a nucleic acid molecule of SEQ ID NO:1; or (d) a polypeptideencoded by a nucleic acid molecule substantially identical to SEQ IDNO:1. A T2R76 polypeptide can also comprise: (a) an isolated nucleicacid molecule encoding a polypeptide of SEQ ID NO:2; (b) an isolatednucleic molecule of SEQ ID NO:1; (c) an isolated nucleic acid moleculewhich hybridizes to a T2R76 nucleic acid sequence under wash stringencyconditions represented by a wash solution having less than about 200 mMsalt concentration and a wash temperature of greater than about 45° C.,and which encodes a T2R76 polypeptide; and (d) an isolated nucleic acidmolecule differing by at least one functionally equivalent codon fromthe isolated nucleic acid molecule of one of (a), (b), and (c) above innucleic acid sequence due to the degeneracy of the genetic code, andwhich encodes a T2R76 polypeptide encoded by the isolated nucleic acidof one of (a), (b), and (c) above.

[0072] The term “substantially identical”, as used herein to describe alevel of similarity between a T2R and a protein substantially identicalthereto, refers to a protein that is at least 35% identical thereto. Forexample, in the case of T2R76 and a protein substantially identical tothis T2R76 protein, this refers to a sequence that is at least about 35%identical to SEQ ID NO:2, when compared over the full length of a T2R76protein. Preferably, a protein substantially identical to a T2R76protein comprises an amino acid sequence that is at least about 35% toabout 45% identical to SEQ ID NO:2, more preferably at least about 45%to about 55% identical to SEQ ID NO:2, even more preferably at leastabout 55% to about 65% identical to SEQ ID NO:2, still more preferablyat least about 65% to about 75% identical to SEQ ID NO:2, still morepreferably at least about 75% to about 85% identical to SEQ ID NO:2,still more preferably at least about 85% to about 95% identical to SEQID NO:2, and still more preferably at least about 95% to about 99%identical to SEQ ID NO:2 when compared over the full length of a T2R76polypeptide. The term “full length” refers to a functional T2R76polypeptide, as described further herein below. Methods for determiningpercent identity between two polypeptides are also defined herein belowunder the heading “Nucleotide and Amino Acid Sequence Comparisons”.

[0073] The term “substantially identical,” when used to describepolypeptides, also encompasses two or more polypeptides sharing aconserved three-dimensional structure. Computational methods can be usedto compare structural representations, and structural models can begenerated and easily tuned to identify similarities around importantactive sites or ligand binding sites. See Saqi et al. (1999)Bioinformatics 15:521-522; Barton (1998) Acta Crystallogr D BiolCrystallogr 54:1139-1146; Henikoff et al. (2000) Electrophoresis21:1700-1706; and Huang et al. (2000) Pac Symp Biocomput:230-241.

[0074] Substantially identical proteins also include proteins comprisingamino acids that are functionally equivalent to amino acids of SEQ IDNO:2. The term “functionally equivalent” in the context of amino acidsis known in the art and is based on the relative similarity of the aminoacid side-chain substituents. See Henikoff & Henikoff (2000) Adv ProteinChem 54:73-97. Relevant factors for consideration include side-chainhydrophobicity, hydrophilicity, charge, and size. For example, arginine,lysine, and histidine are all positively charged residues; that alanine,glycine, and serine are all of similar size; and that phenylalanine,tryptophan, and tyrosine all have a generally similar shape. By thisanalysis, described further herein below, arginine, lysine, andhistidine; alanine, glycine, and serine; and phenylalanine, tryptophan,and tyrosine; are defined herein as biologically functional equivalents.

[0075] In making biologically functional equivalent amino acidsubstitutions, the hydropathic index of amino acids can be considered.Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics, these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine(+2.5); methionine (+1.9); alanine (+1.8); glycine 0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate(−3.5); asparagine (−3.5); lysine (−3.9) and arginine (4.5).

[0076] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982). It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 of the original value is preferred,those which are within ±1 of the original value are particularlypreferred, and those within ±0.5 of the original value are even moreparticularly preferred.

[0077] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 describes that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, e.g., with a biological property of the protein. It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalentprotein.

[0078] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0079] In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those which are within ±1 of theoriginal value are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

[0080] The term “substantially identical” also encompasses polypeptidesthat are biologically functional equivalents of a T2R polypeptide e.g.,T2R76 polypeptide. The term “functional” includes an activity of anT2R76 polypeptide, for example activating intracellular signalingpathways (e.g., coupling with gustducin) and mediating taste perception.Preferably, such activation shows a magnitude and kinetics that aresubstantially similar to that of a cognate T2R polypeptide, e.g., T2R76polypeptide in vivo. Representative methods for assessing T2R76 activityare described herein below.

[0081] The present invention also provides functional fragments of aT2R76 polypeptide. Such functional portion need not comprise all orsubstantially all of the amino acid sequence of a native T2R76 geneproduct.

[0082] The present invention also includes functional polypeptidesequences that are longer sequences than that of a native T2Rpolypeptide e.g., T2R76 polypeptide. For example, one or more aminoacids can be added to the N-terminus or C-terminus of a T2R polypeptidee.g., T2R76 polypeptide. Such additional amino acids can be employed ina variety of applications, including but not limited to purificationapplications. Methods of preparing elongated proteins are known in theart.

[0083] II.C. Nucleotide and Amino Acid Sequence Comparisons

[0084] The terms “identical” or “percent identity” in the context of twoor more nucleotide or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms disclosed herein or by visualinspection.

[0085] The term “substantially identical” in regards to a nucleotide orpolypeptide sequence means that a particular sequence varies from thesequence of a naturally occurring sequence by one or more deletions,substitutions, or additions, the net effect of which is to retain.biological function of a T2R nucleic acid or polypeptide e.g., T2R76nucleic acid or a T2R76 polypeptide.

[0086] For comparison of two or more sequences, typically one sequenceacts as a reference sequence to which one or more test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer program, subsequence coordinatesare designated if necessary, and sequence algorithm program parametersare selected. The sequence comparison algorithm then calculates thepercent sequence identity for the designated test sequence(s) relativeto the reference sequence, based on the selected program parameters.

[0087] Optimal alignment of sequences for comparison can be conducted,for example, by the local homology algorithm of Smith & Waterman (1981)Adv Appl Math 2:482-489, by the homology alignment algorithm ofNeedleman & Wunsch (1970) J Mol Biol 48:443-453, by the search forsimilarity method of Pearson & Lipman (1988) Proc Natl Acad Sci USA85:2444-2448, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, Madison, Wis.), or by visual inspection. Seegenerally, Ausubel (ed.) (1995) Short Protocols in Molecular Biology, 3rd ed. Wiley, New York.

[0088] A preferred algorithm for determining percent sequence identityand sequence similarity is the BLAST algorithm, which is described inAltschul et al. (1990) J Mol Biol 215:403-410.Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of-the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff & Henikoff (11992) Proc Natl Acad Sci USA 89:10915-10919.

[0089] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See e.g., Karlin & Altschul (1993) Proc Natl Acad Sci USA90:5873-5877. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences that would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

[0090] III Methods for Detecting a T2R76 Nucleic Acid

[0091] In another aspect of the invention, a method is provided fordetecting a nucleic acid molecule that encodes a T2R76 polypeptide. Suchmethods can be used to detect T2R76 gene variants or altered geneexpression. For example, detection of a change in T2R76 sequence orexpression can be used for diagnosis of T2R76-related differences intaste perception. Preferably, a nucleic acid used for this methodcomprises the sequence of SEQ ID NO:1.

[0092] Sequences detected by methods of the invention can detected,subcIoned, sequenced, and further evaluated by any measure well known inthe art using any method usually applied to the detection of a specificDNA sequence. Thus, the nucleic acids of the present invention can beused to clone genes and genomic DNA comprising the disclosed sequences.Alternatively, the nucleic acids of the present invention can be used toclone genes and genomic DNA of related sequences. Using the nucleic acidsequences disclosed herein, such methods are known to one skilled in theart. See e.g., Sambrook et al., eds (1989) Molecular Cloning, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Representativemethods are also disclosed in Examples 1-4.

[0093] In one embodiment of the invention, levels of a T2R76 nucleicacid molecule are measured by, for example, using an RT-PCR assay. SeeChiang (1998) J Chromatogr A 806:209-218, and references cited therein.

[0094] In another embodiment of the invention, genetic assays based onnucleic acid molecules of the present invention can be used to screenfor genetic variants, for example by allele-specific oligonucleotide(ASO) probe analysis (Conner et al., 1983), oligonucleotide ligationassays (OLAs) (Nickerson et al., 1990), single-strand conformationpolymorphism (SSCP) analysis (Orita et al., 1989), SSCP/heteroduplexanalysis, enzyme mismatch cleavage, direct sequence analysis ofamplified exons (Kestila et al., 1998; Yuan et al., 1999),allele-specific hybridization (Stoneking et al., 1991), and restrictionanalysis of amplified genomic DNA containing the specific mutation.Automated methods can also be applied to large-scale characterization ofsingle nucleotide polymorphisms (Wang et al., 1998;.Brookes, 1999).Preferred detection methods are non-electrophoretic, including, forexample, the TAQMAN TM allelic discrimination assay, PCR-OLA, molecularbeacons, padlock probes, and well fluorescence. See Landegren et al.(1998) Genome Res 8:769-776 and references cited therein.

[0095] IV. System for Recombinant Expression of a T2R76 Polypeptide

[0096] The present invention further provides a system for expression ofa recombinant T2R76 polypeptide of the present invention. This TR276polypeptide may be expressed with one or more other T2Rs which may behuman or non-human T2Rs. Such a system can be used for subsequentpurification and/or characterization of a T2R76 polypeptide. Forexample, a purified T2R76 polypeptide can be used as an immunogen forthe production of an T2R76 antibody, described further herein below.

[0097] A system for recombinant expression of a T2R76 polypeptide canalso be used for the identification of modulators of a T2R76polypeptide. Alternatively, the disclosed T2R76 polypeptides can be usedas a control polypeptide when assaying the activation of other testpolypeptides. Such test polypeptides can include other T2Rs that areimplicated in taste perception, for example any one of thosepolypeptides disclosed in Adler et al. (2000) Cell 100:693-702 and inMatsunami et al. (2000) Nature 601-603.

[0098] The term “expression system” refers to a host cell comprising aheterologous nucleic acid and the polypeptide encoded by theheterologous nucleic acid. For example, a heterologous expression systemcan comprise a host cell transfected with a construct comprising arecombinant T2R76 nucleic acid, a host cell transfected with T2R76 cRNA,or a cell line produced by introduction of heterologous nucleic acidsinto a host cell genome. As noted, these expression systems may includeother T2R nucleic acids.

[0099] A system for recombinant expression of a T2R76 polypeptide cancomprise: (a) a recombinantly expressed T2R76 polypeptide; and (b) ahost cell comprising the recombinantly expressed T2R76 polypeptide. Forexample, a T2R76 cRNA can be transcribed in vitro and then introducedinto a host cell, whereby a T2R76 polypeptide is expressed. The systemcan further comprise one or more additional T2R polypeptides, in orderto produce a heteromenic T2R receptor comprising hT2R76 and another T2Rpolypeptide..

[0100] A system for recombinant expression of a T2R76 polypeptide canalso comprise: (a) a construct comprising a vector and a nucleic acidmolecule encoding a T2R76 polypeptide operatively linked to aheterologous promoter; and (b) a host cell comprising the construct of(a), whereby the host cell expresses a T2R76 polypeptide. The system canfurther comprise constructs encoding one or more additional T2Rpolypeptides. Additionally, a single construct itself can encode a T2R76polypeptide and one or more additional T2R polypeptides.

[0101] Isolated polypeptides and recombinantly produced polypeptides canbe purified and characterized using a variety of standard techniquesthat are known to the skilled artisan. See e.g., Schröder & Lübke (1965)The Peptides. Academic Press, New York; Schneider & Eberle (1993)Peptides, 1992: Proceedings of the Twenty-Second European PeptideSymposium, Sep. 13-19, 1992, Interlaken, Switzerland. Escom, Leiden;Bodanszky (1993) Principles of Peptide Synthesis, 2 nd rev. ed.Springer-Verlag, Berlin/New York; Ausubel (ed.) (1995) Short Protocolsin Molecular Biolog, 3 rd ed. Wiley, New York.

[0102] Preferably, a recombinantly expressed T2R76 polypeptide comprisesa functional taste receptor, more preferably a bitter taste receptor.Thus, a recombinantly expressed T2R76 polypeptide preferably displaysactivation in response to bitter compounds. Also preferably, arecombinant T2R76 polypeptide shows activation responses similar to anative T2R76 polypeptide. Representative methods for determining T2R76function are described herein below.

[0103] IV.A. Expression Constructs

[0104] A construct for expression of a T2R76 polypeptide includes avector and a T2R76 nucleotide sequence, wherein the T2R76 nucleotidesequence is operatively linked to a promoter sequence. A construct forrecombinant T2R76 expression can also comprise transcription terminationsignals and sequences required for proper translation of the nucleotidesequence. Preparation of an expression construct, including addition oftranslation and termination signal sequences, is known to one skilled inthe art.

[0105] Recombinant production of a T2R polypeptide, e.g., T2R76polypeptide can be directed using a constitutive promoter or aninducible promoter. Representative promoters that can be used inaccordance with the present invention include Simian virus 40 earlypromoter, a long terminal repeat promoter from retrovirus, an actinpromoter, a heat shock promoter, and a metallothien protein.

[0106] Suitable vectors that can be used to express a T2R76 polypeptideinclude but are not limited to viruses such as vaccinia virus oradenovirus, baculovirus vectors, yeast vectors, bacteriophage vectors(e.g., lambda phage), plasmid and cosmid DNA vectors,transposon-mediated transformation vectors, and derivatives thereof.

[0107] Constructs are introduced into a host cell using a transfectionmethod compatible with the vector employed. Standard transfectionmethods include electroporation, DEAE-Dextran transfection, calciumphosphate precipitation, liposome-mediated transfection,transposon-mediated transformation, infection using a retrovirus,particle-mediated gene transfer, hyper-velocity gene transfer, andcombinations thereof.

[0108] IV.B. Host Cells

[0109] The term “host cell”, as used herein, refers to a cell into whicha heterologous nucleic acid molecule can be introduced. Any suitablehost cell can be used, including but not limited to eukaryotic hostssuch as mammalian cells (e.g., HEK-293 cells, HeLa cells, CV-1 cells,COS cells), amphibian cells (e.g., Xenopus oocytes), insect cells (e.g.,Sf9 cells), as well as prokaryotic hosts such as E. coli and Bacillussubtilis. Preferred host cells substantially lack a T2R76 polypeptide.

[0110] A host cell strain can be chosen which modulates the expressionof the recombinant sequence, or modifies and processes the gene productin the specific fashion desired. For example, different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce a non-glycosylated core protein product, andexpression in yeast will produce a glycosylated product.

[0111] The present invention further encompasses recombinant expressionof a T2R76 polypeptide in a stable cell line. Methods for generating astable cell line following transformation of a heterologous constructinto a host cell are known in the art. See e.g., Joyner (1993) GeneTarneting: A Practical Approach. Oxford University Press, Oxford/NewYork. Thus, transformed cells, tissues, or non-human organisms areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny or propagated forms thereof.

[0112] The present invention further encompasses cryopreservation ofcells expressing a recombinant T2R76 polypeptide as disclosed herein.Thus, transiently transfected cells and cells of a stable cell lineexpressing T2R76 can be frozen and stored for later use. Frozen cellscan be readily transported for use at a remote location.

[0113] Cryopreservation media generally consists of a base medium,cryopreservative, and a protein source. The cryopreservative and proteinprotect the cells from the stress of the freeze-thaw process. Forserum-containing medium, a typical cryopreservation medium is preparedas complete medium containing 10% glycerol; complete medium containing10% DMSO (dimethylsulfoxide), or 5.0% cell-conditioned medium with 50%fresh medium with 10% glycerol or 10% DMSO. For serum-free medium,typical cryopreservation formulations include 50% cell-conditioned serumfree medium with 50% fresh serum-free medium containing 7.5% DMSO; orfresh serum-free medium containing 7.5% DMSO and 10% cell culture gradeDMSO. Preferably, a cell suspension comprising about 106 to about 107cells per ml is mixed with cryopreservation medium.

[0114] Cells are combined with cryopreservation medium in a vial orother container suitable for frozen storage, for example NUNC@CRYOTUBESTM (available from Applied Scientific of South San Francisco,Calif.). Cells can also be aliquotted to wells of a multi-well plate,for example a 96-well plate designed for highthroughput assays, andfrozen in plated format.

[0115] Cells are preferably cooled from room temperature to a storagetemperature at a rate of about −1° C. per minute. The cooling rate canbe controlled, for example, by placing vials containing cells in aninsulated water-filled reservoir having about 1 liter liquid capacity,and placing such cube in a −70° C. mechanical freezer. Alternatively,the rate of cell cooling can be controlled at about −1° C. per minute bysubmersing vials in a volume of liquid refrigerant such as an aliphaticalcohol, the volume of liquid refrigerant being more than fifteen timesthe total volume of cell culture to be frozen, and placing the submersedculture vials in a conventional freezer at a temperature below about−70° C. Commercial devices for freezing cells are also available, forexample, the Planer Mini-Freezer R202/200R (Planer Products Ltd. ofGreat Britain) and the BF-5 Biological Freezer (Union CarbideCorporation of Danbury, Conn., United States of America). Preferably,frozen cells are stored at or below about −70° C. to about −80° C., andmore preferably at or below about −130° C.

[0116] To obtain the best possible cell survival, thawing of the cellsmust be performed as quickly as possible. Once a vial, or otherreservoir containing frozen cells is removed from storage, it should beplaced directly into a 37° C. water bath and gently shaken until it iscompletely thawed. If cells are particularly sensitive tocryopreservatives, the cells are centrifuged to remove cryopreservativeprior to further growth.

[0117] Additional methods for preparation and handling of frozen cellscan be found in Freshney (1987) Culture of Animal Cells: A Manual ofBasic Technique. 2 nd ed. A. R. Liss, New York and in U.S. Pat. Nos.6,176,089; 6,140,123; 5,629,145; and 4,455,842; among other places.

[0118] V. Transgenic Animals

[0119] The present invention also provides a transgenic animalcomprising a disruption of T2R76 gene expression and optionally anotherT2R disruptor. Altered gene expression can include expression of analtered level or mutated variant of a T2R76 gene. The present inventionprovides nucleic acids encoding T2R76 that can be used to prepareconstructs for generating a transgenic animal. Also provided is genomiclocalization data useful for preparation of constructs targeted to theT2R 76 locus.

[0120] In one embodiment of the present invention, the transgenic animalcan comprise a mouse with targeted modification of the mouse T2R76 locusand can further comprise mice strains with complete or partialfunctional inactivation of the T2R76 genes in all somatic cells.

[0121] In an alternative embodiment, a transgenic animal in accordancewith the present invention is prepared using anti-sense or ribozymeT2R76 constructs, driven by a universal or tissue-specific promoter, toreduce levels of T2R76 gene expression in somatic cells, thus achievinga “knock-down” phenotype. The present invention also provides thegeneration of murine strains with conditional or inducible inactivationof T2R 76. Such murine strains can also comprise additional synthetic ornaturally occurring mutations, for example a mutation in any other T2Rgene.

[0122] The present invention also provides mice strains with specific“knocked-in” modifications in the T2R76 gene, for example to create anover-expression or dominant negative phenotype. Thus, “knocked-in”modifications include the expression of both wild type and mutated formsof a nucleic acid encoding a T2R76 polypeptide.

[0123] Techniques for the preparation of transgenic animals are known inthe art. Exemplary techniques are described in U.S. Pat. No. 5,489,742(transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396,5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933(transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Pat.No. 5,741,957 (transgenic bovine species), the entire contents of eachof which are herein incorporated by reference.

[0124] For example, a transgenic animal of the present invention cancomprise a mouse with a targeted modification of the mouse T2R76. Micestrains with complete or partial functional inactivation of the T2R76gene in all somatic cells can be generated using standard techniques ofsite-specific recombination in murine embryonic stem cells. See Capecchi(1989) Science 244:1288-1292; Thomas & Capecchi (1990) Nature346:847-850; and Delpire et al. (1999) Nat Genet 22:192195.

[0125] VI. T2R76 Antibodies

[0126] In another aspect of the invention, a method is provided forproducing an antibody that specifically binds a T2R76 polypeptide.According to the method, a full-length recombinant T2R76 polypeptide isformulated so that it can be used as an effective immunogen, and used toimmunize an animal so as to generate an immune response in the animal.The immune response is characterized by the production of antibodiesthat can be collected from the blood serum of the animal. The presentinvention also provides antibodies produced by methods that employ thenovel T2R76 polypeptides disclosed herein, including SEQ ID NO:2.

[0127] The term “antibody” refers to an immunoglobulin protein, orfunctional portion thereof, including a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a hybrid antibody, a singlechain antibody, a mutagenized antibody, a humanized antibody, andantibody fragments that comprise an antigen binding site (e.g., Fab andFv antibody fragments). In a preferred embodiment of the invention, aT2R76 antibody comprises a monoclonal antibody. Thus, the presentinvention also encompasses antibodies and cell lines that producemonoclonal antibodies as described herein.

[0128] The term “specifically binds”, when used to describe binding ofan antibody to a T2R76 polypeptide, refers to binding to a T2R76polypeptide in a heterogeneous mixture of other polypeptides.

[0129] The phrases “substantially lack binding” or “substantially nobinding”, as used herein to describe binding of an antibody to a controlpolypeptide or sample, refers to a level of binding that encompassesnon-specific or background binding, but does not include specificbinding.

[0130] Techniques for preparing and characterizing antibodies are knownin the art. See e.g., Harlow & Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and U.S. Pat. Nos. 4,196,265; 4,946,778; 5,091,513; 5,132,405;5,260,203; 5,677,427; 5,892,019; 5,985,279; and 6,054,561.

[0131] T2R76 antibodies prepared as disclosed herein can be used inmethods known in the art relating to the localization and activity ofT2R76 polypeptides, e.g., for cloning of nucleic acids encoding a T2R76polypeptide, immunopurification of a T2R76 polypeptide, imaging a T2R76polypeptide in a biological sample, and measuring levels of a T2R76polypeptide in appropriate biological samples. To perform such methods,an antibody of the present invention can further comprise a detectablelabel, including but not limited to a radioactive label, a fluorescentlabel, an epitope label, and a label that can be detected in vivo.Methods for selection of a label suitable for a particular detectiontechnique, and methods for conjugating to or otherwise associating adetectable label with an antibody are known to one skilled in the art.

[0132] VIII. T2R76 Modulators

[0133] The present invention further discloses assays to identifymodulators of T2R76 activity. An assay can employ a system forexpression of a T2R76 polypeptide, as disclosed herein above, or anisolated T2R76 polypeptide produced in such a system wherein such T2Rpolypeptide may be expressed with other T2R polypeptides. The presentinvention also provides modulators of T2R76 activity identified usingthe disclosed methods.

[0134] The term “modulate” means an increase, decrease, or otheralteration of any or all chemical and biological activities orproperties of a T2R76 polypeptide. Thus, the method for identifyingmodulators involves assaying a level or quality of T2R76 function.

[0135] A method for identifying a modulator of T2R76 function cancomprise: (a) providing a recombinant expression system whereby a T2R76polypeptide is expressed in a host cell, and wherein the T2R76polypeptide comprises a T2R76 polypeptide; (b) providing a testsubstance to the system of (a); (c) assaying the level or quality ofT2R76 function in the presence of the test substance; (d) comparing thelevel or quality of T2R76 function in the presence of the test substancewith a control level or quality of T2R76 function; and (e) identifying atest substance as a T2R76 modulator by determining a level or quality ofT2R76 function in the presence of the test substance as significantlychanged when compared to a control level or quality of T2R76 function.In some embodiments, the expression system will also provide for T2R76to be co-expressed with at least one other T2R.

[0136] A control level or quality of T2R76 activity refers to a level orquality of wild type T2R76 activity. Preferably, a system forrecombinant expression of a T2R76 polypeptide comprises SEQ ID NO:2.When evaluating the modulating capacity of a test substance, a controllevel or quality of T2R76 activity comprises a level or quality ofactivity in the absence of a test substance.

[0137] The term “significantly changed”, as used herein to refer to analtered level or activity of a T2R polypeptide, e.g., T2R76 polypeptide,and refers to a quantified change in a measurable quality that is largerthan the margin of error inherent in the measurement technique,preferably an increase or decrease by about 2-fold or greater relativeto a control measurement, more preferably an increase or decrease byabout 5-fold or greater, and most preferably an increase or decrease byabout 10-fold or greater.

[0138] In one embodiment of the invention, assaying T2R76 functioncomprises determining a level of T2R76 gene expression.

[0139] In another embodiment of the invention, assaying T2R76 functioncomprises assaying binding activity of a recombinantly expressed T2R76polypeptide. For example, a T2R76 activity can comprise an amount or astrength of binding of a modulator to a T2R76 polypeptide.

[0140] In still another embodiment of the invention, assaying T2R76function can comprise assaying an active conformation of a T2R76polypeptide.

[0141] In a preferred embodiment of the invention, assaying T2R76function comprises assaying activation of intracellular signaling eventsin response to binding of a ligand or a modulator to a T2R76polypeptide. For example, ligand-mediated stimulation of G proteinexchange activity can be assayed by measuring an amount of binding of[³⁵S]GTPγS to a T2R76 polypeptide, as described further herein below andin Example 3.

[0142] Modulators identified by the disclosed methods can compriseagonists and antagonists. As used herein, the term “agonist” means asubstance that activates, synergizes, or potentiates the biologicalactivity of a T2R76 polypeptide. As used herein, the term “antagonist”refers to a substance that blocks or mitigates the biological activityof a T2R76 polypeptide. A modulator can also comprise a ligand or asubstance that specifically binds to a T2R76 polypeptide. Activity andbinding assays for the determination of a T2R76 modulator can beperformed in vitro or in vivo.

[0143] In one embodiment of the invention, such assays are useful forthe identification of T2R76 modulators that can be developed asadditives to alter taste of a composition for oral use, including butnot limited to food, beverages, oral washes, dentifrices, cosmetics, andpharmaceuticals, as described further herein below under the heading“Applications.” For example, an inhibitor of T2R76 can be used to reducebitter taste.

[0144] In another embodiment of the invention, such assays are usefulfor the identification of T2R76 modulators that can be developed asadditives to alter taste of a compound that is of possible butundesirable oral use, for example household cleansers, poisons, etc.Thus, an agonist of T2R76 can be used to introduce or increase bittertaste of a composition to thereby discourage its oral use.

[0145] In still another embodiment of the invention, assays using arecombinant T2R76 polypeptide can be performed for the purpose ofprescreening bioactive agents, wherein an interaction between the agentand T2R76 is undesirable. For example, a drug intended foradministration to a subject can be tested for T2R76 modulating activitythat can result in an undesirable bitter taste.

[0146] In still another embodiment of the invention, an assay disclosedherein can be used to characterize a mutant T2R76 polypeptide, forexample a mutant polypeptide that is linked to a differences in bittertaste perception. Recombinant expression of mutated T2R76 polypeptideswill permit further analysis of disorder-related T2R76 polypeptides.

[0147] In accordance with the present invention there is also provided arapid and high throughput screening method that relies on the methodsdescribed herein. This screening method comprises separately contactinga T2R76 polypeptide with a plurality of test substances. In such ascreening method the plurality of target substances preferably comprisesmore than about 104 samples, or more preferably comprises more thanabout 105 samples, and still more preferably more than about 106samples.

[0148] The in vitro and cellular assays of the invention can comprisesoluble assays, or can further comprise a solid phase substrate forimmobilizing one or more components of the assay. For example, a T2R76polypeptide, or a cell expressing a T2R76 polypeptide, and optionallyanother T2R polypeptide can be bound directly to a solid state componentvia a covalent or non-covalent linkage. Further, optionally, the bindingcan include a linker molecule or tag that mediates indirect binding of aT2R76 polypeptide to a substrate.

[0149] Representative linkers include known binding pairs (e.g., biotinand avidin), antibodies that recognize known antigens, syntheticpolymers (e.g., polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,polyimides, and polyacetates), peptides, ethers. A linker can optionallycomprise a flexible linker, for example ploy (ethelyne glycol) linkers(available from Shearwater Polymers, Inc. of Huntsville, Ala., UnitedStates of America). Optionally, a linker can further comprise amide,sulfhydryl, or heterofunctional binding sites.

[0150] Linkers can be affixed to a solid substrate using any of avariety of current methods, including derivatization of a substratewhereby it reacts with a linker or non-chemical approaches that employheat or ultraviolet cross-linking. Representative protocols can befound, for example, in Merrifield (1963) J Am Chem Soc 85:2149-2154(describing solid phase synthesis of, e.g., peptides); Geysen et al.(11987) J Immun Meth 102:259-274 (describing synthesis of solid phasecomponents on pins); Frank & boring (1988) Tetrahedron 44:60316040(describing synthesis of various peptide sequences on cellulose disks);Fodor et al. (1991) Science 251:767-777; and Kozal et al. (1996) Nat Med2(7):753759 (describing arrays of biopolymers fixed to solidsubstrates), Merrifield (1963) J Am Chem Soc 85:2149-2154 (describingsolid phase synthesis of, e.g., peptides); Geysen et al. (1987) J ImmunMeth 102:259-274 (describing synthesis of solid phase components onpins); Frank & Doring (1988) Tetrahedron 44:60316040 (describingsynthesis of various peptide sequences on cellulose disks); Fodor et al.(1991) Science 251:767-777; and Kozal et al. (1996) Nat Med 2(7):753759(describing arrays of biopolymers fixed to solid substrates), amongother places.

[0151] VII.A. Test Substances

[0152] A potential modulator assayed using the methods of the presentinvention comprises a candidate substance. As used herein, the terms“candidate substance” and “test substance” are used interchangeably, andeach refers to a substance that is suspected to interact with a T2R76polypeptide, including any synthetic, recombinant, or natural product orcomposition. A test substance suspected to interact with a polypeptidecan be evaluated for such an interaction using the methods disclosedherein.

[0153] Representative test substances include but are not limited topeptides, oligomers, nucleic acids (e.g., aptamers), small molecules(e.g., chemical compounds), antibodies or fragments thereof, nucleicacid-protein fusions, any other affinity agent, and combinationsthereof. A test substance can additionally comprise a carbohydrate, avitamin or derivative thereof, a hormone, a neurotransmitter, a virus orreceptor binding domain thereof, an ops or rhodopsin, an odorant, apheromone, a toxin, a growth factor, a platelet activation factor, aneuroactive peptide, or a neurohormone. Preferably, a candidatesubstance elicits bitter taste perception. A candidate substance to betested can be a purified molecule, a homogenous sample, or a mixture ofmolecules or compounds.

[0154] The term “small molecule” as used herein refers to a compound,for example an organic compound, with a molecular weight of less thanabout 1,000 daltons, more preferably less than about 750 daltons, stillmore preferably less than about 600 daltons, and still more preferablyless than about 500 daltons. A small molecule also preferably has acomputed log octanol-water partition coefficient in the range of about−4 to about +14, more preferably in the range of about −2 to about +7.5.

[0155] Test substances can be obtained or prepared as a library. As usedherein, the term “library” means a collection of molecules. A librarycan contain a few or a large number of different molecules, varying fromabout ten molecules to several billion molecules or more. A molecule cancomprise a naturally occurring molecule, a recombinant molecule, or asynthetic molecule. A plurality of test substances in a library can beassayed simultaneously. Optionally, test substances derived fromdifferent libraries can be pooled for simultaneous evaluation.

[0156] Representative libraries include but are not limited to a peptidelibrary (U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409),an oligomer library (U.S. Pat. Nos. 5,650,489 and 5,858,670), an aptamerlibrary (U.S. Pat. Nos. 6,180,348 and 5,756,291), a small moleculelibrary (U.S. Pat. Nos. 6,168,912 and 5,738,996), a library ofantibodies or antibody fragments (U.S. Pat. Nos. 6,174,708, 6,057,098,5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667,988), a library ofnucleic acid-protein fusions (U.S. Pat. No. 6,214,553), and a library ofany other affinity agent that can potentially bind to a T2R76polypeptide (e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538).

[0157] A library can comprise a random collection of molecules.Alternatively, a library can comprise a collection of molecules having abias for a particular sequence, structure, or conformation. See e.g.,U.S. Pat. Nos. 5,264,563 and 5,824,483. Methods for preparing librariescontaining diverse populations of various types of molecules are knownin the art, for example as described in U.S. patents cited herein above.Numerous libraries are also commercially available.

[0158] VII.B. Binding Assays

[0159] In another embodiment of the invention, a method for identifyingof a T2R76 modulator comprises determining specific binding of a testsubstance to a T2R76 polypeptide or a heteromenic receptor comprising aT2R76 polypeptide and one or more other T2R polypeptides. The term“binding” refers to an affinity between two molecules. Preferably,specific binding also encompasses a quality or state of mutual actionsuch that an activity of one protein or compound on another protein isinhibitory (in the case of an inhibitor or antagonist) or enhancing (inthe case of an activator or agonist).

[0160] The phrase “specifically (or selectively) binds”, when referringto the binding capacity of a candidate modulator, refers to a bindingreaction which is determinative of the presence of the protein in aheterogeneous population of proteins and other biological materials. Thebinding of a modulator to a T2R76 polypeptide can be considered specificif the binding affinity is about 1×10⁴M⁻¹ to about 1×10⁶M⁻¹ or greater.The phrase “specifically binds” also refers to saturable binding. Todemonstrate saturable binding of a test substance to a T2R76polypeptide, Scatchard analysis can be carried out as described, forexample, by Mak et al. (1989) J Biol Chem 264:21613-21618.

[0161] The phases “substantially lack binding” or “substantially nobinding”, as used herein to describe binding of a modulator to a controlpolypeptide or sample, refers to a level of binding that encompassesnon-specific or background binding, but does not include specificbinding.

[0162] Several techniques can be used to detect interactions between aT2R76 polypeptide and a test substance without employing a knowncompetitive modulator. Representative methods include, but are notlimited to, Fluorescence Correlation Spectroscopy, Surface-EnhancedLaser Desorption/Ionization Time-Of-flight Spectroscopy, and Biacoretechnology, each technique described herein below. These methods areamenable to automated, high-throughput screening.

[0163] Fluorescence Correlation Spectroscopy (FCS) measures the averagediffusion rate of a fluorescent molecule within a small sample volume(Tallgren, 1980). The sample size can be as low as 103 fluorescentmolecules and the sample volume as low as the cytoplasm of a singlebacterium. The diffusion rate is a function of the mass of the moleculeand decreases as the mass increases. FCS can therefore be applied topolypeptide-ligand interaction analysis by measuring the change in massand therefore in diffusion rate of a molecule upon binding. In a typicalexperiment, the target to be analyzed (e.g., a T2R76 polypeptide) isexpressed as a recombinant polypeptide with a sequence tag, such as apoly-histidine sequence, inserted at the N-terminus or C-terminus. Theexpression is mediated in a host cell, such as E. coli, yeast, Xenopusoocytes, or mammalian cells. The polypeptide is purified usingchromatographic methods. For example, the poly-histidine tag can be usedto bind the expressed polypeptide to a metal chelate column such as Ni²⁺chelated on iminodiacetic acid agarose. The polypeptide is then labeledwith a fluorescent tag such as carboxytetramethylrhoda mine or BODIPYTmreagent (available from Molecular Probes of Eugene, Oreg.). Thepolypeptide is then exposed in solution to the potential ligand, and itsdiffusion rate is determined by FCS using instrumentation available fromCarl Zeiss, Inc. (Thornwood, N.Y.). Ligand binding is determined bychanges in the diffusion rate of the polypeptide.

[0164] Surface-Enhanced Laser Desorption/Ionization (SELDI) wasdeveloped by Hutchens & Yip (1993) Rapid Commun Mass Spectrom7:576-580.When coupled to a time-of-flight mass spectrometer (TOF),SELDI provides a technique to rapidly analyze molecules retained on achip. It can be applied to ligand-protein interaction analysis bycovalently binding the target protein, or portion thereof, on the chipand analyzing by mass spectrometry the small molecules that bind to thisprotein (Worrall et al., 1998). In a typical experiment, a targetpolypeptide (e.g., a T2R76 polypeptide) is recombinantly expressed andpurified. The target polypeptide is bound to a SELDI chip either byutilizing a poly-histidine tag or by other interaction such as ionexchange or hydrophobic interaction. A chip thus prepared is thenexposed to the potential ligand via, for example, a delivery system ableto pipet the ligands in a sequential manner (autosampler). The chip isthen washed in solutions of increasing stringency, for example a seriesof washes with buffer solutions containing an increasing ionic strength.After each wash, the bound material is analyzed by submitting the chipto SELDI-TOF. Ligands that specifically bind a target polypeptide areidentified by the stringency of the w6sh needed to elute them.

[0165] Biacore relies on changes in the refractive index at the surfacelayer upon binding of a ligand to a target polypeptide (e.g., a T2R76polypeptide) immobilized on the layer. In this system, a collection ofsmall ligands is injected sequentially in a 2-5 microliter cell, whereinthe target polypeptide is immobilized within the cell. Binding isdetected by surface plasmon resonance (SPR) by recording laser lightrefracting from the surface. In general, the refractive index change fora given change of mass concentration at the surface layer is practicallythe same for all proteins and peptides, allowing a single method to beapplicable for any protein (Liedberg et al., 1983. In a typicalexperiment, a target protein is recombinantly expressed, purified, andbound to a Biacore chip. Binding can be facilitated by utilizing apoly-histidine tag or by other interaction such as ion exchange* orhydrophobic interaction. A chip thus prepared is then exposed to one ormore potential ligands via the delivery system incorporated in theinstruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in asequential manner (autosampler). The SPR signal on the chip is recordedand changes in the refractive index indicate an interaction between theimmobilized target and the ligand. Analysis of the signal kinetics of onrate and off rate allows the discrimination between non-specific andspecific interaction. See also Homola et al. (1999) Sensors andActuators 54:3-15 and references therein.

[0166] VII.C. Conformational Assay

[0167] The present invention also provides a method for identifying aT2R76 modulator that relies on a conformational change of a T2R76polypeptide expressed alone or in association with another T2Rpolypeptide when bound by or otherwise interacting with a T2R76modulator.

[0168] Application of circular dichroism to solutions of macromoleculesreveals the conformational states of these macromolecules. The techniquecan distinguish random coil, alpha helix, and beta chain conformationalstates.

[0169] To identify modulators of a T2R76 polypeptide, circular dichroismanalysis can be performed using a recombinantly expressed T2R76polypeptide. A T2R76 polypeptide is purified, for example by ionexchange and size exclusion chromatography, and mixed with a testsubstance. The mixture is subjected to circular dichroism. Theconformation of a T2R76 polypeptide in the presence of a test substanceis compared to a conformation of a T2R76 polypeptide in the absence of atest substance. A change in conformational state of a T2R76 polypeptidein the presence of a test substance can thus be used to identify a T2R76modulator. Representative methods are described in U.S. Pat. Nos.5,776,859 and 5,780,242. The T2R76 polypeptide may be comprised in aheteromenic receptor comprising another T2R polypeptide.

[0170] VII.D. Receptor Activation Assays

[0171] In a preferred embodiment of the invention, a method foridentifying a T2R76 modulator employs a functional T2R76 polypeptide.Novel T2R76 polypeptides disclosed herein include SEQ ID NO:2.Representative methods for determining T2R76 function include assayingligand-mediated activation of intracellular signaling events, asdescribed herein below.

[0172] The effect of a test substance on T2R76 function can compriseassaying any physiological change elicited by T2R76 activity, includingbut not limited to phosphorylation of a T2R76 polypeptide, G proteinbinding to a T2R76 polypeptide, ion flux in a cell expressing a T2R76polypeptide, changes in gene transcription, changes in cell metabolism(e.g., cell growth), changes in intracellular second messengers (e.g.,Ca²⁺, IP3, cGMP, cAMP), and changes in transmitter or hormone release.GPCR signal transduction and methods for assaying the same are describedin Methods in Enzymology volumes 237 and 238 (1994). See also Berridge &Irvine (1984) Nature 312:315-321; Bourne et al. (1991) Nature10:349:117-27; Bourne et al. (1990) Nature 348:125-32; Felley-Bosco etal. (1994) Am J Resp Cell and Mol Biol 11:159-164; Mistili & Spector(1997) Nat Biotech 15:961-964; Offermanns & Simon (1995) J Biol Chem270:15175-15180; Pitcher et al. (1998) Annu Rev Biochem 67:653-92; andU.S. Pat. Nos. 4,115,538; 5,436,128; 6,004,808, 6,403,305, and6,255,059.

[0173] In a preferred embodiment of the invention, assaying T2R76function comprises assaying coupling of a recombinantly expressed T2R76polypeptide alone or in association with another T2R polypeptide togustducin or a promiscuous G protein such as Gq or transducin. Arepresentative level of T2R76 activity can thus comprise an amountexchange of GDP for GTPγS on gustducin as described in Example 3. Arepresentative quality of T2R76 activity can comprise, for example, theselective activation of G protein a subunits.

[0174] In accordance with the method, cells expressing T2R76 can beprovided in the form of a kit useful for performing an assay of T2R76function. Thus, cells can be frozen as described herein above andtransported while frozen to others for performance of an assay. Forexample, in one embodiment of the invention, a test kit is provided fordetecting a T2R76 modulator, the kit comprising: (a) frozen cellstransfected with DNA encoding a full-length T2R76 polypeptide; and (b) amedium for growing the cells.

[0175] Preferably, a cell used in such an assay comprises a cell that issubstantially devoid of native T2R76 and polypeptides substantiallysimilar to T2R76. A preferred cell comprises a eukaryotic cell, forexample a HEK-293 cell.

[0176] The term “substantially devoid of”, as used herein to describe ahost cell or a control cell, refers to a quality of having a level ofnative T2R76, a level of a polypeptide substantially similar to T2R76,or a level of activity thereof, comprising a background level. The term“background level” encompasses non-specific measurements of expressionor activity that are typically detected in a cell free of T2R76 and freeof polypeptides substantially similar to a T2R76 polypeptide.

[0177] Cells used in the assays of the invention preferably comprise afunctional G protein that is capable of coupling a T2R76 receptor to anintracellular signaling pathway. In one embodiment of the invention, thefunctional G protein can comprise a G protein that displays promiscuouscoupling, for example Gα15 and Gα16. See Wilkie et al. (1991) Proc AndAcad Sci USA 88:10049-10053 and U.S. Pat. No. 6,004,808.

[0178] Also preferably, all assays employing cells expressingrecombinant T2R76 additionally employ control cells that aresubstantially devoid of native T2R76 and polypeptides substantiallysimilar to a T2R76 polypeptide. When using transiently transfectedcells, a control cell can comprise, for example, an untransfected hostcell. When using a stable cell line expressing a T2R76 polypeptide, acontrol cell can comprise, for example, a parent cell line used toderive the T2R76-expressing cell line.

[0179] Assays of T2R76 activity that employ transiently transfectedcells preferably include a marker that distinguishes transfected cellsfrom non-transfected cells. The term “marker” refers to any detectablemolecule that can be used to distinguish a cell that recombinantlyexpresses T2R76 from a cell that does not recombinantly express a T2R76polypeptide. Preferably, a marker is encoded by or otherwise associatedwith a construct for T2R76 expression, such that cells aresimultaneously transfected with a nucleic acid molecule encoding T2R76and the marker. Representative detectable molecules that are useful asmarkers include but are not limited to a heterologous nucleic acid, apolypeptide encoded by a transfected construct (e.g., an enzyme or afluorescent polypeptide), a binding protein, and an antigen. Forexample, a maker can comprise a rhodopson tag, which can be detectedimmunologically, as described in Example 2.

[0180] Examples of enzymes that are useful as markers includephosphatases (such as acid or alkaline phosphatase), β-galactosidase,urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase,glucoamylase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase,β-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase,alcohol dehydrogenase, or peroxidases (such as horseradish peroxidase).

[0181] A marker comprising an enzyme can be detected based on activityof the enzyme. Thus, a substrate is be added to catalyze a reaction theend product of which is detectable, for example using s pectrophotometer, a luminometer, or a fluorimeter. Substrates for reaction bythe above-mentioned enzymes, and that produce a detectable reactionproduct, are known to one of skill in the art.

[0182] A preferred marker comprises an encoded polypeptide that can bedetected in the absence of an added substrate. Representativepolypeptides that can be detected directly include GFP and EGFP. Commonresearch equipment has been developed to perform high-throughputdetection of fluorescence, for example GFP or EGFP fluorescence,including instruments from GSI Lumonics (Watertown, Mass., United Statesof America), Amersharn Pharmacia Biotech/Molecular Dynamics (Sunnyvale,Calif., United States of America), Applied Precision Inc. (Issauah,Wash., United States of America), and Genomic Solutions Inc. (Ann Arbor,Mich., United States of America). Most of the commercial systems usesome form of scanning technology with photomultiplier tube detection.

[0183] VII. E. Rational Design

[0184] The knowledge of the structure a native T2R76 polypeptideprovides an approach for rational design of modulators and diagnosticagents. In brief, the structure of a T2R76 polypeptide can be determinedby X-ray crystallography and/or by computational algorithms thatgenerate three-dimensional representations. See Saqi et al. (1999)Bioinformatics 15:521-522; Huang et al. (2000) Pac SympBiocomput:230-241; and PCT International Publication No. WO 99/26966.Alternatively, a working model of a T2R76 polypeptide structure can bederived by homology modeling (Maalouf et al., 1998). Computer models canfurther predict binding of a protein structure to various substratemolecules that can be synthesized and tested using the assays describedherein above. Additional compound design techniques are described inU.S. Pat. Nos. 5,834,228 and 5,872,011.

[0185] In general, a T2R76 polypeptide is a membrane protein, and can bepurified in soluble form using detergents or other suitable amphiphilicmolecules. The resulting T2R76 polypeptide is in sufficient purity andconcentration ‘for crystallization. The purified T2R76 polypeptidepreferably runs as a single band under reducing or non-reducingpolyacrylamide gel electrophoresis (PAGE). The purified T2R76polypeptide can be crystallized under varying conditions of at least oneof the following: pH, buffer type, buffer concentration, salt type,polymer type, polymer concentration, other precipitating ligands, andconcentration of purified T2R76. Methods for generating a crystallinepolypeptide are known in the art and can be reasonably adapted fordetermination of a T2R76 polypeptide as disclosed herein. See e.g.,Deisenhofer et al. (1984) J Mol Biol 180:385-398; Weiss et al. (1990)FEBS Lett 267:268-272; or the methods provided in a commercial kit, suchas the CRYSTAL SCREEN™ kit (available from Hampton Research ofRiverside, Calif., United States of America).

[0186] A crystallized T2R76 polypeptide can be tested for functionalactivity and differently sized and shaped crystals are further testedfor suitability in X-ray diffraction. Generally, larger crystals providebetter crystallography than smaller crystals, and thicker crystalsprovide better crystallography than thinner crystals. Preferably, T2R76crystals range in size from 0.1-1.5 mm. These crystals diffract X-raysto at least 10 A resolution, such as 1.5-10.0 A or any range of valuetherein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 A orless being preferred for the highest resolution.

[0187] VIII. Methods for Detecting a T2R76 Polypeptide

[0188] The present invention further provides methods for detecting aT2R76 polypeptide. The disclosed methods can be used for determiningaltered levels of T2R76 expression that are associated withT2R76-related differences in taste perception.

[0189] In one embodiment of the invention, the method involvesperforming an immunochernical reaction with an antibody thatspecifically recognizes a T2R76 polypeptide, wherein the antibody wasprepared according to a method of the present invention for producingsuch an antibody. Thus, the method comprises: (a) obtaining a biologicalsample comprising peptidic material; (b) contacting the biologicalsample with an antibody that specifically binds a T2R76 polypeptide andthat was produced according to the disclosed methods, wherein theantibody comprises a detectable label; and (c) detecting the detectablelabel, whereby a T2R76 polypeptide in a sample is detected.

[0190] Techniques for detecting such antibody-antigen conjugates orcomplexes are known in the art and include but are not limited tocentrifugation, affinity chromatography and other immunochernicalmethods. See e.g., Manson (1992) Immunochemical Protocols. Humana Press,Totowa, N.J., United States of America; Ishikawa (1999) Ultrasensitiveand Rapid Enzyme Immunoassa . Elsevier, Amsterdam/New York, UnitedStates of America; Law (1996) Immunoassay: Practical Guide. Taylor &Francis, London/Bristol, Pa., United States of America; Chan (1996)Immunoassay Automation: An Updated Guide to Systems. Academic Press, SanDiego; Liddell & Weeks (1995) Antibody Technology. Bios ScientificPublishers, Oxford, United Kingdom; Masseyeff et al. (1993) Methods ofImmunological Analysis. VCH Verlagsgesellschaft/VCH Publishers,Weinheim, Federal Republic of Germany/New York, United States ofAmerica; Walker & Rapley (1993) Molecular and Antibody Probes inDiagnosis. Wiley, Chichester, N.Y.; Wyckoff et al. (1985) DiffractionMethods for Biological Macromolecules. Academic Press, Orlando, Fla.,United States of America; and references cited therein.

[0191] In another embodiment of the invention, a modulator that showsspecific binding to a T2R76 polypeptide is used to detect a T2R76polypeptide. Analogous to detection of a T2R76 polypeptide using anantibody, the method comprises: (a) obtaining a biological samplecomprising peptidic material; (b) contacting the biological sample witha modulator of a T2R76 polypeptide, wherein the modulator comprises adetectable label; and (c) detecting the detectable label, whereby aT2R76 polypeptide in a sample is detected. Any suitable detectable labelcan be used, for example a fluorophore or epitope label.

[0192] IX. Applications

[0193] The present invention provides methods for identification ofmodulators of a T2R76 polypeptide. The modulators of the invention areuseful for altering bitter taste perception, for example to suppress orenhance bitter taste perception.

[0194] IX.A. Subjects

[0195] The term “subject” as used herein includes any vertebratespecies, preferably warm-blooded vertebrates such as mammals and birds.More particularly, the methods of the present invention are contemplatedfor the treatment of tumors in mammals such as humans, as well as thosemammals of importance due to being endangered (such as Siberian tigers),of economical importance (animals raised on farms for consumption byhumans) and/or social importance (animals kept as pets or in zoos) tohumans, for instance, carnivores other than humans (such as cats anddogs), swine (pigs, hogs, and wild boars), ruminants and livestock (suchas cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thosekinds of birds that are endangered or kept in zoos, as well as fowl, andmore particularly domesticated fowl or poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomical importance to humans.

[0196] IX.B. Compositions

[0197] In accordance with the methods of the present invention, acomposition that is administered to alter taste perception in a subjectcomprises an effective amount of a T2R76 modulator. A T2R76 modulatorcan comprise any one of the types of test substances described hereinabove. T2R76 modulators identified as disclosed herein can be used toprepare a composition for oral use, including but not limited to food,beverages, oral washes, dentifrices, cosmetics, and pharmaceuticals, forexample any of those compound listed herein below. T2R76 modulators canalso be used as additives to alter the taste of a compound that is ofpossible but undesirable oral use, for example household cleansers,poisons, etc.

[0198] Representative foods having an undesirable or bitter tasteinclude, but are not limited to, citrus fruits such as grapefruit,orange, and lemon; vegetables such as tomato, pimento, celery, melon,carrot, potato, and asparagus; seasoning or flavoring materials such asflavor, sauces, soy sauce, and red pepper; foods originating fromsoybean; emulsion foods such as cream, dressing, mayonnaise, andmargarine; processed marine products such as fish meat, ground fishmeat, and fish eggs; nuts such as peanuts; fermented foods such asfermented soybean; meats and processed meats; pickles; noodles; soupsincluding powdery soups; dairy products such as cheese; breads andcakes; confectioneries such as candies, chewing gum, and chocolate; andspecifically prepared foods for health.

[0199] Representative cosmetics eliciting bitter taste (e.g., skinlotions, creams, face packs, lip sticks, foundations, shavingpreparations, after-shave lotions, cleansing foams, and cleansing gels)include but are not limited to those compositions that includesurfactants such as sodium alkyl sulfate and sodium monoalkyl phosphate;fragrances such as menthol, linalool, phenylethyl alcohol, ethylpropionate, geraniol, linalyl acetate and benzyl acetate; antimicrobialssuch as methyl paraben, propyl paraben and butyl paraben; humectantssuch as lactic acid and sodium lactate; alcohol-denaturating agents suchas sucrose octaacetate and brucine; and astringents such as aluminumlactate.

[0200] Representative pharmaceuticals having a bitter taste includeacetaminophen, terfenadine, guaifenesin, trimethoprim, prednisolone,ibuprofen, prednisolone sodium phosphate, methacholine, neostigmine,epinephrine, albuterol, pseudoephedrine hydrochloride, diphenhydramine,chlorpheniramine maleate, phenothiazine, chlorpromazine, ch lordiazepoxide, amitriptyline, barbiturates, diphenylhydantoin, caffeine,morphine, demerol, codeine, lomotil, lidocaine, salicylic acid,sulfonamides, chloroquine, a vitamin preparation, minerals andpenicillins.

[0201] The modulators can also be administered as part of prepared food,beverage, oral wash, dentifrice, cosmetic, or drug. To prepare acomposition for administration to a subject, a T2R76 modulator can beadmixed with a compound whose taste is to be modulated in amountcomprising about 0.001% to about 10% by weight, preferably from about0.01% to about 8% by weight, more preferably from about 0.1% to about 5%by weight, and most preferably from about 0.5% to about 2% by weight.

[0202] Suitable formulations include solutions, extracts, elixirs,spirits, syrups, suspensions, powders, granules, capsules, pellets,tablets, and aerosols. Optionally, a formulation can include apharmaceutically acceptable carrier, a suspending agent, a solubilizer,a thickening agent, a stabilizer, a preservative, a flavor, a colorant,a sweetener, a perfume, or a combination thereof. T2R76 modulators andcompositions can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials.

[0203] IX.C. Administration

[0204] T2R76 modulators can be administered directly to a subject formodulation of taste perception. Preferably, a modulator of the inventionis administered orally or nasally.

[0205] In accordance with the methods of the present invention, aneffective amount of a T2R76 modulator is administered to a subject. Theterm “effective amount” refers to an amount of a composition sufficientto modulate T2R76 activation and/or to modulate bitter taste perception.

[0206] An effective amount can be varied so as to administer an amountof an T2R76 modulator that is effective to achieve the desired tasteperception. The selected dosage level will depend upon a variety offactors including the activity of the T2R76 modulator, formulation,combination with other compositions (e.g., food, drugs, etc.), theintended use (e.g., as a food additive, dentifrice, etc.), and thephysical condition and prior medical history of the subject beingtreated.

[0207] An effective amount or dose can be readily determined using invivo assays of taste perception as are known in the art. Representativemethods for assaying taste perception are described in Example 4.

EXAMPLES

[0208] The following Examples have been included to illustrate modes ofthe invention. Certain aspects of the following Examples are describedin terms of techniques and procedures found or contemplated by thepresent co-inventors to work well in the practice of the invention.These Examples illustrate standard laboratory practices of theco-inventors. In light of the present disclosure and the general levelof skill in the art, those of skill will appreciate that the followingExamples are intended to be exemplary only and that. numerous changes,modifications, and alterations can be employed without departing fromthe scope of the invention.

Example 1

[0209] Cloning of Human T2R76.

[0210] A novel gene encoding a human bitter taste receptor wasidentified in the human genome sequence databases. Novel hT2R member,hT2R76 is located on human chromosome 7. The chromosomal location ofT2R76 DNA sequence was determined by screening the University ofCalifornia (Santa Cruz, Calif.) Genomics web site. This analysis showedthat T2R76 is located on chromosome 7 in the region 144062692-144063648.The bitter taste of certain compounds, such as phenylthiocarbarnate,have been linkage genetically to chromosomes 5 and 7. (Guo et al. (2001)Ann Hum Biol 28:111-42). Thus, T2R76 is predicted to be involved inbinding and recognition of certain bitter tastants.

[0211] Human T2R76 was initially identified by reiterated sequencesearch of DNA sequence databases with previously described hT2Rsequences. A full-length open reading frame encoding hT2R76 was thenisolated by PCR amplification of genomic DNA. The amino acid sequence ofhT2R76 was derived by conceptual translation of the corresponding openreading frame. The hT2R76 nucleotide and amino acid sequences are setforth as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0212] The intronless open reading frame of hT2R76 encodes a putativereceptor protein 318 amino acid residues in length. A comparison of thehT2R76 protein sequence with all known proteins in the public sequencedatabases using the BLASTP algorithm revealed its strong homology to themembers of the mammalian bitter receptor family.

Example 2

[0213] Construction of rhod-hT2R76.

[0214] A bridge overlap PCR extension technique was used to generaterhod-hT2R76 chimeras, which contain the first 38 amino acids of bovinerhodopsin in frame with human T2R76 coding sequences as describedChandrashekar et al. (2000) Cell 100:703-711. The chimeric rhod-hT2R76gene was then cloned into the pFastBac-1 vector, and baculovirusescontaining rhodopsin-tagged hT2R76 was produced using Bac-to-Bac system(Invitrogen Corporation of Carlsbad, Calif., United States of America).Expression of hT2R76 was confirmed by immunoblotting usinganti-rhodopsin tag antibodies (136-30). Sf9 cells infected withhT2R76encoding baculovirus produced a protein of the expected molecularweight (−35 kDa).

Example 3

[0215] In Vitro G Protein Coupling of T2R76.

[0216] An infectious bacmid encoding rhod-hT2R76 is prepared asdescribed in Example 2. Insect larval cells are infected for 60 hourswith recombinant bacmid and membranes are prepared as described by Ryba& Trindelli (1995) J Biol Chem 270:6757-6767. Peripheral proteins areremoved by treatment with 5M urea and membranes are resuspended in 1 OmMHEPES pH 7.5, 1 mM EDTA, and 1 mM DTT. The expression of rhod-hT2R76 canbe assessed by Western blotting using monocolonal antibody B6-30.

[0217] G proteins are isolated, for example as described by Hoon et al.(1995) Cell 96 629-636 and by Ryba & Trindelli (1995) J Biol Chem270:6757-6767. Receptor-catalyzed exchange of GDP for GTPγS on gustducinis measured in the presence of 10 nM rhod-hT2R76, 100 μtM GDP, and 20 μMGβ1γ8. GDP-GTPγS exchange on promiscuous G proteins (e.g., Gα5 ortransducin) is performed as described in U.S. patent application Ser.No. 60/372,089. Measurements made at about 15-60 minute time pointsreflect the initial rate of GTPγS binding.

Example 4

[0218] Taste Study.

[0219] A flavor acceptance study is conducted using a test compositioncomprising a T2R76 modulator identified as disclosed herein. A controlcomposition lacking the T2R76 modulator, but which is otherwisesubstantially similar or identical to the test composition, is alsoused. The study employs a two-way crossover design, with all subjectsevaluating both compositions, which are administered in one or more sameamounts or doses. The test and control compositions are evaluated on asingle study day. The sequence for administering the test and controlcompositions is randomized among subjects. All enrolled subjectscomplete all aspects of the study protocol. Subjects respond to each ofthe test and control compositions using ordinal taste scores (e.g.,1=very bitter, 2=bitter, 3=indifferent, 4=not that bitter, 5=not bitterat all). Adverse events are recorded. Effectiveness of a T2R76 modulatoris determined by measuring a significant difference in palatability ofthe test composition when compared to the control composition.

Example 5

[0220] Response of hT2R76 to Bitter Compounds.

[0221] A GTPγS binding assay is effected using a mammalian cell line(HEK293) that expresses hT2R76 as well as a control cell line thatexpresses a different hT2R (hT2R64). These cell lines are contacted withbitter compounds including 6-n-propylthiouracil (PROP), sucroseoctaacetate, raffinose undacaacetate, (RUA), copper glycinate,denatonium and quinine at different concentrations ranging from 0.5 to 2mm. The results of this assay are used to confirm that hT2R76 is abitter taste receptor that is specifically activated by known bittertaste stimuli. In this GTPγS binding assay activity is determined eitherin the presence or absence of specific concentrations of known bittercompounds.

Example 6

[0222] High Throughput Screening Assay

[0223] Using the GTPγS binding assay, a library of over 15,000 compoundsis screened to identify other compounds that specifically activatehT2R76. The structure of the specific compounds that activate hT276 inthis assay are compared in order to predict compounds having similarstructure that potentially will activate hT2R76. Libraries of compoundshaving these similar structures are then evaluated at differentconcentrations in the same GTPγS binding assay to identify othercompounds that activate hT2R76.

Example 7

[0224] Human Taste Test.

[0225] The compounds which activate hT2R76 in GTPγS binding assays areevaluated in human taste tests. These human taste tests are performed inconsenting adults who are orally administered the identified compound atthe concentration at which they activate hT2R76 in vitro. In these tastetests an identified compound (which activates hT2R76) is dissolved inwater to achieve a compound concentration that activates hT2R76 in thein vitro GTPγS binding assay.

[0226] In this taste test, a sample of at least 5 persons taste a seriesof aqueous solutions containing a bitter compound. (In the preferredexample, the bitter compound is a T2R76 agonist). Each of the personsranks the degree of bitterness in a labeled magnitude scale ranging from0 to 100 (0 is “barely detectable.” and 100 is “strongest imaginable”).Next, each person tastes a series of aqueous solutions containing thebitter compound and the T2R76 inhibitor and ranks the degree ofbitterness for each sample. The effictiveness of the T2R76 inhibitor ismeasured by the reduction in the degree of bitterness. As a means ofcomparison, a known bitter compound (quinine sulfate) is also tested andevaluated by each subject. The result of the taste tests are representedas the average rating in all subjects.

Example 8

[0227] Response of hT2R76 to Known Bitter Compounds

[0228] The results of this assay are used to identify bitter cmopoundsthat activate hT2R76. Based thereon, assays can be developed thatidentify compounds that block the activation of hT2R76 by such bittercompounds.

Conclusion

[0229] The results of these assays will provide a demonstration that theGTPγS binding assay can be used to identity bitter compounds and thathT2R761 functions as a human bitter taste receptor. The identifiedcompounds can be used to provide bitterness to foods and beverages.Alternatively, these compounds can be used as agonists in assays for theidentification of bitter blockers and modulators and other bittercompounds.

[0230] References

[0231] The references listed below as well as all references cited inthe specification are incorporated herein by reference to the extentthat they supplement, explain, provide a background for or teachmethodology, techniques and/or compositions employed herein.

[0232] Adler E, Hoon M A, Mueller K L, Chandrashekar J, Ryba N J, ZukerC S (2000) A novel family of mammalian taste receptors. Cell 100(6):693-702.

[0233] Altschul S F, Gish W, Miller W, Myers E W & Lipman D J (1990)Basic Local Alignment Search Tool. J Mol Biol 215:403-410.

[0234] Ausubel F, ed (1995) Short Protocols in Molecular Biolog , 3 rded. Wiley, New York.

[0235] Barton G J (1998) Protein Sequence Alignment Techniques. ActaCrystallogr D Biol Crystallogr 54:1139-1146.

[0236] Bateman A, Birney E, Durbin R, Eddy S R, Howe K L & Sonnhammer EL (2000) The PFAM Protein Families Database. Nucleic Acids Res28:263-266.

[0237] Batzer M A, Carlton J E & Deininger P L (1991) EnhancedEvolutionary PCR Using Oligonucleotides with Inosine at the 3′-Terminus.Nucleic Acids Res 19:5081.

[0238] Berridge & Irvine (1984) Inositol trisphosphate, a novel secondmessenger in cellular signal transduction. Nature 312:315-321.

[0239] Bodanszky M (1993) Principles of Peptide Synthesis, 2 nd rev. ed.Springer-Verlag, Berlin/New York.

[0240] Bourne H R, Sanders D A & McCormick F (1990) The GTPasesuperfamily: a conserved switch for diverse cell functions. Nature348:125-132.

[0241] Bourne H R, Sanders D A & McCormick F (1991) Nature The GTPasesuperfamily: conserved structure and molecular mechanism. 349:117-127.

[0242] Brookes A J (1999) The Essence of SNPs. Gene 234:177-186.

[0243] Burge C & Karlin S (1997) Prediction of Complete Gene Structuresin Human Genomic DNA. J Mol Biol 268:78-94.

[0244] Burge C B & Karlin S (1998) Finding the Genes in Genomic DNA.Curr Opin Struct Biol 8:346-354.

[0245] Capecchi M R (1989a) Altering the Genome by HomologousRecombination. Science 244:1288-1292.

[0246] Capecchi M R (1989b) Altering the Genome by HomologousRecombination. Science 244:1288-1292.

[0247] Chan D W (1996) Immunoassay Automation: An Updated Guide toSystems. Academic Press, San Diego, Calif., United States of America.

[0248] Chandrashekar J, Mueller K L, Hoon M A, Adler E, Feng L, Guo W,Zuker C S, Ryba N J (2000) T2Rs function as bitter taste receptors. Cell100(6): 703-711.

[0249] Chiang L W (1998) Detection of Gene Expression in Single Neuronsby Patch-Clamp and Single-Cell Reverse Transcriptase Polymerase ChainReaction. J Chromatogr A 806:209-218.

[0250] Conner B J, Reyes A A, Morin C, Itakura K, Teplitz R L & WallaceR B (1983) Detection of Sickle Cell Beta S-Globin Allele byHybridization with Synthetic Oligonucleotides. Proc Natl Acad Sci USA80:278-282.

[0251] Costanzi E, Beccari T, Stinchi S, Bibi L, Hopwood J J & OrlacchioA (2000) Gene Encoding the Mouse Sulphamidase: cDNA Cloning, Structure,and Chromosomal Mapping. Mamm Genome 11:436-439.

[0252] Deisenhofer J, Epp 0, Miki K, Huber R & Michel H (1984) X-RayStructure Analysis of a Membrane Protein Complex. Electron Density Mapat 3 a Resolution and a Model of the Chromophores of the PhotosyntheticReaction Center from Rhodopseudomonas Viridis. J Mol Biol 180:385-398.

[0253] Felley-Bosco E, Ambs S, Lowenstein C J, Keefer L K & Harris C C(1994) Constitutive expression of inducible nitric oxide synthase inhuman bronchial epithelial cells induces c-fos and stimulates the cGMPpathway. Am J Resp Cell and Mol Biol 11: 159-164.

[0254] Fewell J G, MacLaughlin F, Mehta V, Gondo M, Nicol F, Wilson E &Smith L C (2001) Gene Therapy for the Treatment of Hemophilia B UsingPINC Formulated Plasmid Delivered to Muscle with Electroporation. MolTher 3:574-583.

[0255] Frank & Doring (1988) Simultaneous Multiple Peptide SynthesisUnder Continuous Flow Conditions On Cellulose Paper Discs As SegmentalSolid Supports. Tetrahedron 44:60316040.

[0256] Freshney R I (1987) Culture of Animal Cells: A Manual of BasicTechnique, 2 nd ed. A. R. Liss, New York.

[0257] Geysen H M, Rodda S J, Mason T J, Tribbick G & Schoofs P G (1987)Strategies for epitope analysis using peptide synthesis. J Immun Meth102:259-274.

[0258] Glover D M & Hames B D (1995) DNA Cloning: A Practical Approach,2 nd ed. IRL Press at Oxford University Press, Oxford/New York.

[0259] Guo S W & Reed D R (2001) The genetics of phenylthiocarbamideperception. Ann Hum Biol 28:111-142.

[0260] Harlow E & Lane D (1988) Antibodies: A Laboratory Manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., United Statesof America.

[0261] Henikoff J G, Pietrokovski S, McCallum C M & Henikoff S (2000)Blocks-Based Methods for Detecting Protein Homology. Electrophoresis21:1700-1706.

[0262] Henikoff S & Henikoff J G (1992) Amino Acid Substitution Matricesfrom Protein Blocks. Proc Natl Acad Sci, USA 89:10915-10919.

[0263] Henikoff S & Henikoff J G (2000) Amino Acid SubstitutionMatrices. Adv Protein Chem 54:73-97.

[0264] Homola J, Yee S & Gauglitz G (1999) Surface Plasmone ResonanceSensors: Review. Sensors and Actuators B 54:3-15.

[0265] Hoon M A, Adler E, Lindemeier J, Battey J F, Ryba N J P & Zuker CS (1995) “Functional expression of the taste specific G protein,alpha-gustducin.” Cell 96629-636.

[0266] Huang C C, Novak W R, Babbitt P C, Jewett Al, Ferrin T E & KleinT E (2000) Integrated Tools for Structural and Sequence Alignment andAnalysis. Pac Symp Biocomput:230-241.

[0267] Hutchens & Yip (1993) New Desorption Strategies for the MassSpectrometric Analysis of Macromolecules. Rapid Communications in MassSpectroscopy 7:576-580.

[0268] Ishikawa E (1999) Ultrasensitive and Rapid Enzyme Immunoassa .Elsevier, Amsterdam/New York.

[0269] Jayaraman S, Teitler L, Skalski B & Verkman A S (1999)Long-Wavelength lodide-Sensitive Fluorescent Indicators for Measurementof Functional CFTR Expression in Cells. Am J Physiol 277:Cl 008-1018.

[0270] Joyner A L (1993) Gene Targeting: A Practical Approach. OxfordUniversity Press, Oxford/New York.

[0271] Karlin S & Altschul S F (1993) Applications and Statistics forMultiple High-Scoring Segments in Molecular Sequences. Proc Natl AcadSci, USA 90:5873-5877.

[0272] Kozal M J, Shah N, Shen N, Yang R, Fucini R, Merigan T C, RichmanD D, Morris D, Hubbell E, Chee M & Gingeras T R (1996) Extensivepolymorphisms observed in HIV-1 clade B protease gene using high-densityoligonucleotide arrays. Nat Med 2(7):753-759.

[0273] Kyte J & Doolittle R F (1982) A Simple Method for Displaying theHydropathic Character of a Protein. J Mol Biol 157:105-132.

[0274] Landegren U, Nilsson M & Kwok P Y (1998) Reading Bits of GeneticInformation: Methods for Single-Nucleotide Polymorphism Analysis. GenomeRes 8:769-776. Law (1996) Immunoassay: A Practical Guide. Taylor &Francis, London/Bristol, Pa., United States of America.

[0275] Lindemann B. (2001) Receptors and transduction in taste. Nature413(6852): 219-225.

[0276] Maalouf G J, Xu W, Smith T F & Mohr S C (1998) Homology Model forthe Ligand-Binding Domain of the Human Estrogen Receptor. J BiomolStruct Dyn 15:841-851.

[0277] Mak P, McDonnell D P, Weigel N L, Schrader W T & O'Malley B W(1989) Expression of Functional Chicken Oviduct Progesterone Receptorsin Yeast (Saccharomyces Cerevisiae). J Biol Chem 264:21613-21618.

[0278] Manson M M (1992) Immunochernical Protocols. Humana Press,Totowa, N.J., United States of America Masseyeff R F, Albert W H W &Staines N (1993) Methods of Immunological Analysis.

[0279] V C H Verlagsgesellschaft; VCH Publishers, Weinheim (FederalRepublic of Germany)/New York, N.Y. (United States of America).

[0280] Matsunami H, Montmpyeur J P, Buck L B (2000) A family ofcandidate taste receptors in human and mouse. Nature 404(6778): 601-604.

[0281] Merrifield (1963) J Am Chem Soc 85:2149-2154.

[0282] Mistili & Spector (1997) Nat Biotech 15:961-964.

[0283] Needleman S B & Wunsch C D (1970) A General Method Applicable tothe Search for Similarities in the Amino Acid Sequence of Two Proteins.J Mol Biol 48:443453.

[0284] Nickerson D A, Kaiser R, Lappin S, Stewart J, Hood L & LandegrenU (1990) Automated DNA Diagnostics Using an ELISA-Based OligonucleotideLigation Assay. Proc Natl. Acad Sci, USA 87:8923-8927.

[0285] Offermanns S & Simon M I (1995) G alpha 15 and G alpha 16 couplea wide variety of receptors to phospholipase C. J Biol Chem270:15175-15180.

[0286] Ohtsuka E, Matsuki S, Ikehara M, Takahashi Y & Matsubara K (1985)An Alternative Approach to Deoxyoligonucleotides as Hybridization Probesby Insertion of Deoxyinosine at Ambiguous Codon Positions. J Biol Chem260:2605-2608.

[0287] Orita M, Iwahana H, Kanazawa H, Hayashi K & Sekiya T (1989)Detection of Polymorphisms of Human DNA by Gel Electrophoresis asSingle-Strand Conformation Polymorphisms. Proc Natl Acad Sci, USA86:2766-2770.

[0288] PCT International Publication No. WO 99/26966

[0289] PCT International Publication No. WO 01/18050

[0290] PCT International Publication No. WO 01/77676

[0291] Pitcher A Freedman N J & Lefkowitz R J (1998) G protein-coupledreceptor kinases. Annu Rev Biochem 67:653-92.

[0292] Quandt K, Frech K, Karas H, Wingender E & Werner T (1995) Matindand Matinspector: New Fast and Versatile Tools for Detection ofConsensus Matches in Nucleotide Sequence Data. Nucleic Acids Res23:4878-4884.

[0293] Roberts L (1991) GRAIL Seeks out Genes Buried in DNA Sequence.Science 254:805.

[0294] Rossolini G M, Cresti S, Ingianni A, Cattani P, Riccio M L &Satta G (1994) Use of Deoxyinosine-Containing Primers Vs DegeneratePrimers for Polymerase Chain Reaction Based on Ambiguous SequenceInformation. Mol Cell Probes 8:91-98.

[0295] Ryba N J P & Trindelli R (1995) “A novel GTP-binding proteingamma-subunit, G gamma 8, is expressed during neurogenesis in theolfactory and vorneronasal neuroepithelia.” J Biol Chem 270:6757-6767

[0296] Sambrook J, Sambrook E F & Maniatis F (1989) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., United States of America.

[0297] Saqi M A, Wild D L & Hartshorn M J (1999) Protein Analyst—aDistributed Object Environment for Protein Sequence and StructureAnalysis. Bioinformatics 15:521-522.

[0298] Schneider C H & Eberle A N (1993) Pentides, 1992: Proceedings ofthe Twenty-Second European Peptide Symposium, Sep. 13-19, 1992.Interlaken Switzerland. Escom, Leiden.

[0299] Schröder E & Lübke K (1965) The Peptides. Academic Press, NewYork.

[0300] Silhavy T J, Berman M L, Enquist L W & Cold Spring HarborLaboratory. (1984) Experiments with Gene Fusions. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., United States of America.

[0301] Smith T F & Waterman M (1981) Comparison of Biosequences. AdvAppl Math 2:482-489.

[0302] Stoneking M, Hedgecock D, Higuchi R G, Vigilant L & Erlich H A(1991) Population Variation of Human mtDNA Control Region SequencesDetected by Enzymatic Amplification and Sequence-SpecificOligonucleotide Probes. Am J Hum Genet 48:370-382.

[0303] Taylor G, Vimr E, Garman E & Laver G (1992) Purification,Crystallization and Preliminary Crystallographic Study of Neuraminidasefrom Vibrio Cholerae and Salmonella Typhimurfum Lt2. J Mol Biol226:1287-1290.

[0304] Tijssen (1993) Laboratory Technigues in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes. Elsevier, NewYork.

[0305] U.S. Pat. No. 4,115,538

[0306] U.S. Pat. No. 4,196,265

[0307] U.S. Pat. No. 4,455,842

[0308] U.S. Pat. No. 4,554,101

[0309] U.S. Pat. No. 4,736,866

[0310] U.S. Pat. No. 4,946,778

[0311] U.S. Pat. No. 5,091,513

[0312] U.S. Pat. No. 5,132,405

[0313] U.S. Pat. No. 5,162,215

[0314] U.S. Pat. No. 5,223,409

[0315] U.S. Pat. No. 5,260,203

[0316] U.S. Pat. No. 5,264,563

[0317] U.S. Pat. No. 5,436,128

[0318] U.S. Pat. No. 5,489,742

[0319] U.S. Pat. No. 5,498,538

[0320] U.S. Pat. No. 5,550,316

[0321] U.S. Pat. No. 5,573,933

[0322] U.S. Pat. No. 5,629,145

[0323] U.S. Pat. No. 5,614,396

[0324] U.S. Pat. No. 5,625,125

[0325] U.S. Pat. No. 5,648,061

[0326] U.S. Pat. No. 5,650,489

[0327] U.S. Pat. No. 5,667,988

[0328] U.S. Pat. No. 5,677,427

[0329] U.S. Pat. No. 5,702,892

[0330] U.S. Pat. No. 5,741,957

[0331] U.S. Pat. No. 5,738,996

[0332] U.S. Pat. No. 5,747,334

[0333] U.S. Pat. No. 5,756,291

[0334] U.S. Pat. No. 5,776,859

[0335] U.S. Pat. No. 5,780,225

[0336] U.S. Pat. No. 5,780,242

[0337] U.S. Pat. No. 5,824,483

[0338] U.S. Pat. No. 5,834,228

[0339] U.S. Pat. No. 5,840,479

[0340] U.S. Pat. No. 5,858,670

[0341] U.S. Pat. No. 5,872,011

[0342] U.S. Pat. No. 5,892,019

[0343] U.S. Pat. No. 5,922,254

[0344] U.S. Pat. No. 5,948,635

[0345] U.S. Pat. No. 6,004,808

[0346] U.S. Pat. No. 6,057,098

[0347] U.S. Pat. No. 6,107,059

[0348] U.S. Pat. No. 6,140,123

[0349] U.S. Pat. No. 6,156,511

[0350] U.S. Pat. No. 6,168,912

[0351] U.S. Pat. No. 6,174,708

[0352] U.S. Pat. No. 6,176,089

[0353] U.S. Pat. No. 6,180,348

[0354] U.S. Pat. No. 6,190,700

[0355] U.S. Pat. No. 6,214,553

[0356] U.S. Pat. No. 6,255,059

[0357] U.S. Pat. No. 6,403,305

[0358] Walker M R & Rapley R (1993) Molecular and Antibody Probes inDiagnosis. Wiley,

[0359] Chichester/N.Y.

[0360] Wang D G, Fan J B, Siao C J, Berno A, Young P, Sapolsky R,Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L,Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris M S,Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson T J, Lander E S &et al. (1998) Large-Scale Identification, Mapping, and Genotyping ofSingle-Nucleotide Polymorphisms in the Human Genome. Science280:1077-1082.

[0361] Weiss M S, Wacker T, Weckesser J, Welte W & Schulz G E (1990) TheThree-Dimensional Structure of Porin from Rhodobacter Capsulatus at 3 aResolution. FEBS Lett 267:268-272.

[0362] Wilkie T M, Scherle P A, Strathmann M P, Slepak V Z & Simon M I(1991) Characterization of G-protein alpha subunits in the Gq class:expression in murine tissues and in stromal and hernatopoietic celllines. Proc Natl Acad Sci, USA 88:10049-10053.

[0363] Wong G T, Gannon K S, Margolskee R F (1996) Transduction ofbitter and sweet taste by gustducin. Nature 381(6585): 796-800.

[0364] Worrall T A, Cotter R J & Woods A S (1998) Purification ofContaminated Peptides and Proteins on Synthetic Membrane Surfaces forMatrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal Chem70:750-756.

[0365] Wyckoff H W, Hirs C H W & Timasheff S N (1985) DiffractionMethods for Biological Macromolecules. Academic Press, Orlando, Fla.,United States of America.

[0366] Yuan B, Thomas J P, von Kodolitsch Y & Pyeritz R E (1999)Comparison of Heteroduplex Analysis, Direct Sequencing, and EnzymeMismatch Cleavage for Detecting Mutations in a Large Gene, FBN1. HumMutat 14:440-446.

[0367] It will be understood that various details of the invention canbe changed without departing from the scope of the invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation—the inventionbeing defined by the claims appended hereto.

1 2 1 957 DNA Homo sapiens CDS (1)..(954) 1 atg aat gga gac cac atg gttcta gga tct tcg gtg act gac aag aag 48 Met Asn Gly Asp His Met Val LeuGly Ser Ser Val Thr Asp Lys Lys 1 5 10 15 gcc atc atc ttg gtt acc atttta ctc ctt tta cgc ctg gta gca ata 96 Ala Ile Ile Leu Val Thr Ile LeuLeu Leu Leu Arg Leu Val Ala Ile 20 25 30 gca ggc aat ggc ttc atc act gctgct ctg ggc gtg gag tgg gtg cta 144 Ala Gly Asn Gly Phe Ile Thr Ala AlaLeu Gly Val Glu Trp Val Leu 35 40 45 cgg aga atg ttg ttg cct tgt gat aagtta ttg gtt agc cta ggg gcc 192 Arg Arg Met Leu Leu Pro Cys Asp Lys LeuLeu Val Ser Leu Gly Ala 50 55 60 tct cgc ttc tgt ctg cag tca gtg gta atgggt aag acc att tat gtt 240 Ser Arg Phe Cys Leu Gln Ser Val Val Met GlyLys Thr Ile Tyr Val 65 70 75 80 ttc ttg cat ccg atg gcc ttc cca tac aaccct gta ctg cag ttt cta 288 Phe Leu His Pro Met Ala Phe Pro Tyr Asn ProVal Leu Gln Phe Leu 85 90 95 gct ttc cag tgg gac ttc ctg aat gct gcc acctta tgg tcc tct acc 336 Ala Phe Gln Trp Asp Phe Leu Asn Ala Ala Thr LeuTrp Ser Ser Thr 100 105 110 tgg ctc agt gtc ttc tat tgt gtg aaa att gctacc ttc acc cac cct 384 Trp Leu Ser Val Phe Tyr Cys Val Lys Ile Ala ThrPhe Thr His Pro 115 120 125 gtc ttc ttc tgg cta aag cac aag ttg tct gggtgg cta cca tgg atg 432 Val Phe Phe Trp Leu Lys His Lys Leu Ser Gly TrpLeu Pro Trp Met 130 135 140 ctc ttc agc tct gta ggg ctc tcc agc ttc accacc att cta ttt ttc 480 Leu Phe Ser Ser Val Gly Leu Ser Ser Phe Thr ThrIle Leu Phe Phe 145 150 155 160 ata ggc aac cac aga atg tat cag aac tattta agg aac cat cta caa 528 Ile Gly Asn His Arg Met Tyr Gln Asn Tyr LeuArg Asn His Leu Gln 165 170 175 cct tgg aat gtc act ggc gat agc ata cggagc tac tgt gag aaa ttc 576 Pro Trp Asn Val Thr Gly Asp Ser Ile Arg SerTyr Cys Glu Lys Phe 180 185 190 tat ctc ttc cct cta aaa atg att act tggaca atg ccc act gct gtc 624 Tyr Leu Phe Pro Leu Lys Met Ile Thr Trp ThrMet Pro Thr Ala Val 195 200 205 ttt ttc att tgc atg att ttg ctc atc acatct ctg gga aga cac agg 672 Phe Phe Ile Cys Met Ile Leu Leu Ile Thr SerLeu Gly Arg His Arg 210 215 220 aag aag gct ctc ctt aca acc tca gga ttccga gag ccc agt gtg cag 720 Lys Lys Ala Leu Leu Thr Thr Ser Gly Phe ArgGlu Pro Ser Val Gln 225 230 235 240 gca cac ata aag gct ctg ctg gct ctcctc tct ttt gcc atg ctc ttc 768 Ala His Ile Lys Ala Leu Leu Ala Leu LeuSer Phe Ala Met Leu Phe 245 250 255 atc tca tat ttc ctg tca ctg gtg ttcagt gct gca ggt att ttt cca 816 Ile Ser Tyr Phe Leu Ser Leu Val Phe SerAla Ala Gly Ile Phe Pro 260 265 270 cct ctg gac ttt aaa ttc tgg gtg tgggag tca gtg att tat ctg tgt 864 Pro Leu Asp Phe Lys Phe Trp Val Trp GluSer Val Ile Tyr Leu Cys 275 280 285 gca gca gtt cac ccc atc att ctg ctcttc agc aac tgc agg ctg aga 912 Ala Ala Val His Pro Ile Ile Leu Leu PheSer Asn Cys Arg Leu Arg 290 295 300 gct gtg ctg aag agt cgy cgt tcc tcaagg tgt ggg aca cct tga 957 Ala Val Leu Lys Ser Arg Arg Ser Ser Arg CysGly Thr Pro 305 310 315 2 318 PRT Homo sapiens 2 Met Asn Gly Asp His MetVal Leu Gly Ser Ser Val Thr Asp Lys Lys 1 5 10 15 Ala Ile Ile Leu ValThr Ile Leu Leu Leu Leu Arg Leu Val Ala Ile 20 25 30 Ala Gly Asn Gly PheIle Thr Ala Ala Leu Gly Val Glu Trp Val Leu 35 40 45 Arg Arg Met Leu LeuPro Cys Asp Lys Leu Leu Val Ser Leu Gly Ala 50 55 60 Ser Arg Phe Cys LeuGln Ser Val Val Met Gly Lys Thr Ile Tyr Val 65 70 75 80 Phe Leu His ProMet Ala Phe Pro Tyr Asn Pro Val Leu Gln Phe Leu 85 90 95 Ala Phe Gln TrpAsp Phe Leu Asn Ala Ala Thr Leu Trp Ser Ser Thr 100 105 110 Trp Leu SerVal Phe Tyr Cys Val Lys Ile Ala Thr Phe Thr His Pro 115 120 125 Val PhePhe Trp Leu Lys His Lys Leu Ser Gly Trp Leu Pro Trp Met 130 135 140 LeuPhe Ser Ser Val Gly Leu Ser Ser Phe Thr Thr Ile Leu Phe Phe 145 150 155160 Ile Gly Asn His Arg Met Tyr Gln Asn Tyr Leu Arg Asn His Leu Gln 165170 175 Pro Trp Asn Val Thr Gly Asp Ser Ile Arg Ser Tyr Cys Glu Lys Phe180 185 190 Tyr Leu Phe Pro Leu Lys Met Ile Thr Trp Thr Met Pro Thr AlaVal 195 200 205 Phe Phe Ile Cys Met Ile Leu Leu Ile Thr Ser Leu Gly ArgHis Arg 210 215 220 Lys Lys Ala Leu Leu Thr Thr Ser Gly Phe Arg Glu ProSer Val Gln 225 230 235 240 Ala His Ile Lys Ala Leu Leu Ala Leu Leu SerPhe Ala Met Leu Phe 245 250 255 Ile Ser Tyr Phe Leu Ser Leu Val Phe SerAla Ala Gly Ile Phe Pro 260 265 270 Pro Leu Asp Phe Lys Phe Trp Val TrpGlu Ser Val Ile Tyr Leu Cys 275 280 285 Ala Ala Val His Pro Ile Ile LeuLeu Phe Ser Asn Cys Arg Leu Arg 290 295 300 Ala Val Leu Lys Ser Arg ArgSer Ser Arg Cys Gly Thr Pro 305 310 315

What is claimed is:
 1. An isolated T2R76 nucleic acid moleculecomprising: (a) an isolated nucleic acid molecule encoding a polypeptideof SEQ ID NO:2; (b) an isolated nucleic acid molecule of SEQ ID NO:1; or(c) an isolated nucleic acid molecule substantially similar to SEQ IDNO:1.
 2. The isolated T2R76 nucleic acid molecule of claim 1 selectedfrom the group consisting of: (a) an isolated nucleic acid moleculeencoding a polypeptide of SEQ ID NO:2; (b) an isolated nucleic acidmolecule of SEQ ID NO:1; (c) an isolated nucleic acid molecule whichhybridizes to a nucleic acid sequence of SEQ ID NO: 1 under washstringency conditions represented by a wash solution having less thanabout 200 mM salt concentration and a wash temperature of greater thanabout 45° C., and which encodes a T2R76 polypeptide; and (d) an isolatednucleic acid molecule differing by at least one functionally equivalentcodon from the isolated nucleic acid molecule of one of (a), (b), and(c) above in nucleic acid sequence due to the degeneracy of the geneticcode, and which encodes a T2R76 polypeptide encoded by the isolatednucleic acid of one of (a), (b), and (c) above.
 3. The isolated T2R76nucleic acid molecule of claim 1 comprising: (a) an isolated nucleicacid molecule encoding a polypeptide of SEQ ID NO:2; or (b) an isolatednucleic acid molecule of SEQ ID NO:1.
 4. A method for detecting a T2R76nucleic acid molecule, the method comprising: (a) procuring a biologicalsample having nucleic acid material; (b) hybridizing an isolated T2R76nucleic acid molecule under stringent hybridization conditions to thebiological sample of (a), thereby forming a duplex structure between theisolated T2R76 nucleic acid and a nucleic acid within the biologicalsample; and (c) detecting the duplex structure of (b), whereby a T2R76nucleic acid molecule in the biological sample is detected.
 5. Anisolated T2R76 polypeptide comprising: (a) a polypeptide of SEQ ID NO:2;(b) a polypeptide substantially identical to SEQ ID NO:2; (c) apolypeptide encoded by a nucleic acid molecule of SEQ ID NO:1; or (d) apolypeptide encoded by a nucleic acid molecule substantially identicalto SEQ ID NO:1.
 6. The isolated T2R76 polypeptide of claim 5, furthercomprising a polypeptide encoded by a nucleic acid molecule selectedfrom the group consisting of: (a) an isolated nucleic acid moleculeencoding a polypeptide of SEQ ID NO:2; (b) an isolated nucleic moleculeof SEQ ID NO:1; (c) an isolated nucleic acid molecule which hybridizesto a T2R 76 nucleic acid sequence under wash stringency conditionsrepresented by a wash solution having less than about 200 mM saltconcentration and a wash temperature of greater than about 45° C., andwhich encodes a T2R76 polypeptide; and (d) an isolated nucleic acidmolecule differing by at least one functionally equivalent codon fromthe isolated nucleic acid molecule of one of (a), (b), and (c) above innucleic acid sequence due to the degeneracy of the genetic code, andwhich encodes a T2R76 polypeptide encoded by the isolated nucleic acidof one of (a), (b), and (c) above.
 7. The isolated T2R76 polypeptide ofclaim 5 comprising SEQ ID NO:2.
 8. An isolated T2R polypeptide accodingto claim 5 which is associated with at least one other T2R polypeptide.9. The isolated T2R polypeptide of claim 8 wherein said other T2Rpolypeptide is another human T2R.
 10. The isolated T2R polypeptide ofclaim 9 wherein said other human T2R is selected from the groupconsisting of human T2R51, T2R54, T2R55, T2461. T2R63, T2R64, T2R65,T2R67, T2R71, T2R75, T2R59 and T2R33.
 11. A method for producing anantibody that specifically recognizes a T2R76 the isolated nucleic acidof one of (a), (b), and (c) above.
 12. The method of claim 10, whereinthe isolated T2R76 polypeptide comprises SEQ ID NO:2.
 13. The method ofclaim 8, further comprising preparing a monoclonal antibody.
 14. Anantibody produced by the method of claim
 11. 15. A method for detectinga level of a T2R76 polypeptide, the method comprising (a) obtaining abiological sample having peptidic material; (b) detecting a T2R76polypeptide in the biological sample of (a) by immunochemical reactionwith the antibody of claim 14, whereby an amount of T2R76 polypeptide ina sample is determined.
 16. A system for heterologous expression of aT2R76 polypeptide comprising: (a) a T2R76 polypeptide; and (b) aheterologous host cell expressing the T2R76 polypeptide.
 17. The systemof claim 16, wherein the T2R76 polypeptide comprises: (a) a polypeptideof SEQ ID NO:2; (b) a polypeptide substantially identical to SEQ IDNO:2; (c) a polypeptide encoded by a nucleic acid molecule of SEQ IDNO:1; or (d) a polypeptide encoded by a nucleic acid moleculesubstantially identical to SEQ ID NO:1.
 18. The system of claim 17,wherein the T2R76 polypeptide further comprises a polypeptide encoded bya nucleic acid molecule selected from the group consisting of: (a) anisolated nucleic acid molecule encoding a polypeptide of SEQ ID NO:2;(b) an isolated nucleic molecule of SEQ ID NO:1; (c) an isolated nucleicacid molecule which hybridizes to a T2R76 nucleic acid sequence underwash stringency conditions represented by a wash solution having lessthan about 200 mM salt concentration and a wash temperature of greaterthan about 45° C., and which encodes a T2R76 polypeptide; and (d) anisolated nucleic acid molecule differing by at least one functionallyequivalent codon from the isolated nucleic acid molecule of one of (a),(b), and (c) above in nucleic acid sequence due to the degeneracy of thegenetic code, and which encodes a T2R76 polypeptide encoded by theisolated nucleic acid of one of (a), (b), and (c) above.
 19. The systemof claim 18, wherein the isolated T2R76 polypeptide comprises SEQ IDNO:2.
 20. The system of claim 18 which further comprises a nucleic acidencoding another T2R.
 21. The system of claim 16, wherein the host cellcomprises a mammalian cell.
 22. The system of claim 21, wherein themammalian cell comprises a human cell.
 23. The system of claim 16,wherein the host cell further comprises a G protein alpha subunitcapable of coupling to a T2R76 polypeptide.
 24. The system of claim 23,wherein the G protein alpha subunit comprises a promiscuous G protein.25. The system of claim 24, wherein the promiscuous G protein comprisesGα15.
 26. The system of claim 24, wherein the promiscuous G proteincomprises transducin or gustducin.
 27. A method for identifying amodulator of a T2R76 polypeptide, the method comprising: (a) providing arecombinant expression system whereby a T2R76 polypeptide is expressedin a heterologous host cell alone or in combination with at least oneother T2R polypeptide, (b) providing a test substance to the system of(a); (c) assaying a level or quality of T2R76 function in the presenceof the test substance; (d) comparing the level or quality of T2R76function in the presence of the test substance with a control level orquality of T2R76 function; and (e) identifying a test substance as aT2R76 modulator by determining a level or quality of T2R76 function inthe presence of the test substance as significantly changed whencompared to a control level or quality of T2R76 function.
 28. The methodof claim 27, wherein the T2R76 polypeptide comprises: (a) a polypeptideof SEQ ID NO:2; (b) a polypeptide substantially identical to SEQ IDNO:2; (c) a polypeptide encoded by a nucleic acid molecule of SEQ IDNO:1; or (d) a polypeptide encoded by a nucleic acid moleculesubstantially identical to SEQ ID NO:1.
 29. The method of claim 28,wherein the T2R76 polypeptide further comprises a polypeptide encoded bya nucleic acid molecule selected from the group consisting of: (a) anisolated nucleic acid molecule encoding a polypeptide of SEQ ID NO:2;(b) an isolated nucleic molecule of SEQ ID NO: 1; (c) an isolatednucleic acid molecule which hybridizes to a T2R76 nucleic acid sequenceunder wash stringency conditions represented by a wash solution havingless than about 200 mM salt concentration and a wash temperature ofgreater than about 45° C., and which encodes a T2R76 polypeptide; and(d) an isolated nucleic acid molecule differing by at least onefunctionally equivalent codon from the isolated nucleic acid molecule ofone of (a), (b), and (c) above in nucleic acid sequence due to thedegeneracy of the genetic code, and which encodes a T2R76 polypeptideencoded by the isolated nucleic acid of one of (a), (b), and (c) above.30. The method of claim 29, wherein the isolated T2R76 polypeptidecomprises SEQ ID NO:2.
 31. The method of claim 27, wherein the host cellcomprises a mammalian cell.
 32. The method of claim 31, wherein themammalian cell comprises a human cell.
 33. The method of claim 27,wherein the host cell further comprises a G protein alpha subunitcapable of coupling to a T2R76 polypeptide.
 34. The method of claim 33,wherein the G protein alpha subunit comprises a promiscuous G protein.35. The method of claim 34, wherein the promiscuous G protein comprisesGα15.
 36. The method of claim 34, wherein the promiscuous G proteincomprises transducin.
 37. The method of claim 27, wherein the assayingcomprises determining an amount of GTPγS binding.
 38. A T2R76 modulatoridentified by the method of claim
 27. 39. The T2R76 modulator of claim38, further comprising a modulator selected from the group consisting ofa protein, a peptide, an antibody, a nucleic acid, and a small molecule.40. A method for modulating bitter taste perception in a subject, themethod comprising: (a) preparing a composition comprising a modulator ofclaim 38; (b) administering an effective dose of the composition to asubject, whereby bitter taste perception is altered in the subject. 41.The method of claim 40, wherein the composition further comprises afood, a beverage, an oral wash, a dentifrice, a cosmetic, or apharmaceutical.
 42. The method of claim 40, further comprisingco-administering the composition comprising a modulator and acomposition selected from the group consisting of a food, a beverage, anoral wash, a dentifrice, a cosmetic, and a pharmaceutical.
 43. Themethod of claim 40, wherein the subject is a mammal.
 44. The method ofclaim 43, wherein the mammal is a human.
 45. A method for identifyingmodulator of a T2R76 polypeptide, the method comprising: (a) exposing aT2R76 polypeptide alone or a T2R76 polypeptide expressed in associationwith at least one other T2R polypeptide to one or more test substances;(b) assaying binding of a test substance to the isolated T2R76polypeptide or a T2R76 polypeptide combination; and (c) selecting acandidate substance that demonstrates specific binding to the T2R76polypeptide.
 46. The method of claim 45, wherein the T2R76 polypeptidecomprises: (a) a polypeptide of SEQ ID NO:2; (b) a polypeptidesubstantially identical to SEQ ID NO:2; (c) a polypeptide encoded by anucleic acid molecule of SEQ ID NO:1; or (d) a polypeptide encoded by anucleic acid molecule substantially identical to SEQ ID NO:1.
 47. Themethod of claim 46, wherein the T2R76 polypeptide further comprises apolypeptide encoded by a nucleic acid molecule selected from the groupconsisting of: (a) an isolated nucleic acid molecule encoding apolypeptide of SEQ ID NO:2; (b) an isolated nucleic molecule of SEQ IDNO:1; (c) an isolated nucleic acid molecule which hybridizes to a T2R76nucleic acid sequence under wash stringency conditions represented by awash solution having less than about 200 mM salt concentration and awash temperature of greater than about 45° C., and which encodes a T2R76polypeptide; and (d) an isolated nucleic acid molecule differing by atleast one functionally equivalent codon from the isolated nucleic acidmolecule of one of (a), (b), and (c) above in nucleic acid sequence dueto the degeneracy of the genetic code, and which encodes a T2R76polypeptide encoded by the isolated nucleic acid of one of (a), (b), and(c) above.
 48. The method of claim 47, wherein the isolated T2R76polypeptide comprises SEQ ID NO:2.
 49. A T2R76 modulator identified bythe method of claim
 48. 50. The T2R76 modulator of claim 49, furthercomprising a modulator selected from the group consisting of a protein,a peptide, an antibody, a nucleic acid, and a small molecule.
 51. Amethod for modulating bitter taste perception in a subject, the methodcomprising: (a) preparing a composition comprising a modulator of claim49; (b) administering an effective dose of the composition to a subject,whereby bitter taste perception is altered in the subject.
 52. Themethod of claim 51, wherein the composition further comprises a food, abeverage, an oral wash, a dentifrice, a cosmetic, or a pharmaceutical.53. The method of claim 51, further comprising co-administering thecomposition comprising a modulator and a composition selected from thegroup consisting of a food, a beverage, an oral wash, a dentifrice, acosmetic, and a pharmaceutical.
 54. The method of claim 51, wherein theT2R76 modulator is selected from the group consisting of a protein, apeptide, an antibody, a nucleic acid, and a small molecule.
 55. Themethod of claim 51, wherein the subject is a mammal.
 56. The method ofclaim 55, wherein the mammal is a human.
 57. A method for reducingbitter taste perception of a bitter compound, the method comprisingco-administering a T2R76 inhibitor and the bitter compound to a subject.58. The method of claim 57, wherein the co-administering comprisesadministering a composition comprising the T2R76 inhibitor admixed withthe bitter compound.
 59. The method of claim 57, wherein the T2R76inhibitor further comprises a modulator selected from the groupconsisting of a protein, a peptide, an antibody, a nucleic acid, and asmall molecule.
 60. The method of claim 57, wherein the bitter compoundcomprises a food, a beverage, an oral wash, a dentifrice, a cosmetic, ora pharmaceutical.
 61. The method of claim 57, wherein the subject is amammal.
 62. The method of claim 61, wherein the mammal is a human.
 63. Amethod for enhancing bitter taste perception of a compound, the methodcomprising co-administering a T2R76 agonist and the compound to asubject.
 64. The method of claim 63, wherein the co-administeringcomprises administering a composition comprising the T2R76 agonistadmixed with the compound.
 65. The method of claim 63, wherein the T2R76agonist further comprises a modulator selected from the group consistingof a protein, a peptide, an antibody, a nucleic acid, and a smallmolecule.
 66. The method of claim
 63. wherein the subject is a mammal.67. The method of claim 66, wherein the mammal is a human.